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Небесная энциклопедия

Космические корабли и станции, автоматические КА и методы их проектирования, бортовые комплексы управления, системы и средства жизнеобеспечения, особенности технологии производства ракетно-космических систем

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Мониторинг СМИ

Мониторинг СМИ и социальных сетей. Сканирование интернета, новостных сайтов, специализированных контентных площадок на базе мессенджеров. Гибкие настройки фильтров и первоначальных источников.

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05-01-2012 дата публикации

Isolation of Biomolecules from Biological Samples

Номер: US20120003710A1

Автор: Barbara Dawn Leinweber, Paul E. Diggins

Принадлежит: Individual

Nanoparticles for use in the collection, concentration, isolation and storage of biomolecules from biological samples are provided. More specifically, nanoparticles used to isolate biomolecules, including nucleic acids and proteins, cells, cell fragments, bacteria, and viruses from biological samples such as urine, cerebrospinal fluid (CSF), mouthwash samples, and amniotic fluid are provided. Kits for using nanoparticles for the isolation of biomolecules are also provided.

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Номер записи: 1

11-04-2019 дата публикации

Дозированная форма для выделения днк

Номер: RU0000188424U1

Автор: Важа Хутаевич Пация, Вахтанг Почхуа, Гиоргий Жгенти, Марина Почхуа, Мераб Георгиевич Чикобава, Олег Васильевич Рубальский, Ульрих Мартин

Принадлежит: Важа Хутаевич Пация, Вахтанг Почхуа, Гиоргий Жгенти, Марина Почхуа, Мераб Георгиевич Чикобава, Олег Васильевич Рубальский, Ульрих Мартин

Полезная модель относится к медицине и биологии и предназначена для использования при производстве наборов для выделения ДНК, применяемых при проведении молекулярно-генетических исследований. Дозированная форма для выделения ДНК включает ядро, содержащее совместно спрессованные мочевину, натрия хлорид и диоксид кремния, а также сплошную оболочку, состоящую, по меньшей мере, из одного гидрофобного ингредиента, выбранного из ряда: парафин и воск, и выполнена в виде глобулы или таблетки, при этом глобула имеет предпочтительный диаметр 2,0-4,0 мм, а таблетка имеет предпочтительный диаметр 2,0-4,0 мм и предпочтительную толщину 2,0-4,0 мм, причем содержание мочевины, натрия хлорида и диоксида кремния в ядре составляет, мас. %: мочевина - 67,5-80,0, натрия хлорид - 10,0-25,5 и диоксид кремния - 7,0-10,0. Техническим результатом полезной модели является увеличение срока хранения дозированной формы, предотвращение перекрестной контаминации реакционных смесей и обеспечение их пароизоляции. РОССИЙСКАЯ ФЕДЕРАЦИЯ (19) RU (11) (13) 188 424 U1 (51) МПК C12M 3/02 (2006.01) C12N 15/10 (2006.01) C12Q 1/68 (2006.01) ФЕДЕРАЛЬНАЯ СЛУЖБА ПО ИНТЕЛЛЕКТУАЛЬНОЙ СОБСТВЕННОСТИ (12) ОПИСАНИЕ ПОЛЕЗНОЙ МОДЕЛИ К ПАТЕНТУ (52) СПК C12M 3/02 (2019.02); C12N 15/1003 (2019.02); C12Q 1/68 (2019.02) (21) (22) Заявка: 2018128273, 02.08.2018 (24) Дата начала отсчета срока действия патента: 02.08.2018 11.04.2019 (45) Опубликовано: 11.04.2019 Бюл. № 11 Адрес для переписки: 414004, г. Астрахань, ул. Софьи Перовской, 64, кв. 56, Рубальскому Олегу Васильевичу R U 1 8 8 4 2 4 U 1 (56) Список документов, цитированных в отчете о поиске: US 2017/0081704 A1, 23.03.2017. US 5405951 A, 11.04.1995. RU 2539030 C1, 10.01.2015. RU 2116795 C1, 10.08.1998. BY 15648 C1, 30.04.2012. (54) ДОЗИРОВАННАЯ ФОРМА ДЛЯ ВЫДЕЛЕНИЯ ДНК (57) Реферат: Полезная модель относится к медицине и предпочтительный диаметр 2,0-4,0 мм, а таблетка биологии и предназначена для использования при имеет предпочтительный диаметр 2,0-4,0 мм и производстве ...

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Номер записи: 2

31-01-2022 дата публикации

Устройство для выделения и очистки нуклеиновых кислот и/или макромолекул из биологического образца

Номер: RU0000209043U1

Автор: Александр Николаевич Неваленный, Анна Леонидовна Ясенявская, Гиоргий Александрович Ростошвили, Игорь Юрьевич Алексанян, Кристина Шотаевна Арнаудова, Любовь Михайловна Титова, Марина Александровна Самотруева, Мария Романовна Копылова, Ольга Александровна Башкина, Юрий Александрович Максименко

Принадлежит: Федеральное государственное бюджетное образовательное учреждение высшего образования "Астраханский государственный медицинский университет" Министерства здравоохранения Российской Федерации (ФГБОУ ВО Астраханский ГМУ Минздрава России), Федеральное государственное бюджетное образовательное учреждение высшего образования Астраханский государственный технический университет, ФГБОУ ВО "АГТУ"

Полезная модель относится к области молекулярной биологии, биотехнологии и биохимии и может быть использована в медицине. Полезная модель представляет собой устройство для выделения и очистки магнитных наночастиц при экстракции и очистке нуклеиновых кислот (ДНК и РНК) сорбентным методом из различного биологического материала.Технический результат - повышение эффективности работы устройства при его использовании.Устройство имеет верхнюю и нижнюю пластины, держатель пробирок с ручками для переноски, расположенный на верхней пластине, который имеет отверстия и оптические окна, неподвижно закрепленные на нижней пластине вертикальные стойки, оснащенные фиксаторами для ограничения движения по ним в вертикальной плоскости верхней пластины, на стойках жестко фиксированы две горизонтальные оси, с горизонтальными осями связана движущаяся по ним направляющая с жестко закрепленными на ней магнитами в форме стержней, каждый магнит снабжен жестко закрепленной на нем защитной крышкой из пластикового коррозионностойкого материала, держатель пробирок выполнен из отдельных сегментов, имеющих один ряд отверстий для пробирок. РОССИЙСКАЯ ФЕДЕРАЦИЯ (19) RU (11) (13) 209 043 U1 (51) МПК C12M 1/00 (2006.01) C12N 15/10 (2006.01) ФЕДЕРАЛЬНАЯ СЛУЖБА ПО ИНТЕЛЛЕКТУАЛЬНОЙ СОБСТВЕННОСТИ (12) ОПИСАНИЕ ПОЛЕЗНОЙ МОДЕЛИ К ПАТЕНТУ (52) СПК C12M 1/00 (2021.08); C12N 15/10 (2021.08) (21)(22) Заявка: 2021125218, 25.08.2021 (24) Дата начала отсчета срока действия патента: Дата регистрации: 31.01.2022 (45) Опубликовано: 31.01.2022 Бюл. № 4 2 0 9 0 4 3 R U (73) Патентообладатель(и): Федеральное государственное бюджетное образовательное учреждение высшего образования Астраханский государственный технический университет, ФГБОУ ВО "АГТУ" (RU), Федеральное государственное бюджетное образовательное учреждение высшего образования "Астраханский государственный медицинский университет" Министерства здравоохранения Российской Федерации (ФГБОУ ВО Астраханский ГМУ Минздрава России) (RU) (56) Список документов, ...

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Номер записи: 3

12-01-2012 дата публикации

Synthesis of tagged nucleic acids

Номер: US20120009649A1

Автор: Gary Dahl, Roy Rabindranauth Sooknanan

Принадлежит: Cellscript Inc

The present invention relates generally to methods, compositions and kits for synthesizing sense RNA molecules from one or more RNA molecules of interest in a sample. In exemplary embodiments, the methods use a terminal tagging oligoribonucleotide (rTTO) to join a DNA sequence tag to the 3′-termini of first-strand cDNA molecules. The use of an rTTO comprising ribonucleotides results in decreased oligonucleotide-derived background synthesis of RNA in the absence of sample RNA and, surprisingly and unexpectedly, also results in significantly increased yields of sense RNA molecules that exhibit sequences that are substantially identical to those of the RNA molecules of interest in the sample. The sense RNA molecules also have an RNA sequence tag on their 5′-termini that is useful for fixing the lengths of sense RNA molecules that are synthesized in a second or subsequent round.

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Номер записи: 4

19-01-2012 дата публикации

Methods for generation of rna and (poly)peptide libraries and their use

Номер: US20120015840A1

Автор: Michael Lebens

Принадлежит: Individual

The present invention relates to a method for generating an RNA library or a (poly)peptide library comprising the steps of: (a) providing one or more nucleic acid molecules each comprising i) two or more coding elements (A) each giving rise to an RNA molecule upon transcription and/or a (poly)peptide upon transcription and translation; and ii) linking elements (B) arranged according to the general formula of B(AB) 2+n , wherein said linking elements comprise one or more sequence motifs not found in said two or more coding elements allowing specific disruption of the linking elements (B); (b) cloning the nucleic acid molecule of step (a) into a vector; (c) transforming a host cell with the vector obtained in step (b) and propagating said transformed cell; (d) preparing vector DNA from the transformed and propagated cells of step (c); (e) (i) disrupting the vector DNA obtained in step (d) with one or more agents recognizing said one or more sequence motifs of the linking elements or (ii) performing an amplification step with the vector DNA obtained in step (d) and primers hybridizing to the sequence of said linking elements so that the sequences comprising the coding elements (A) are specifically amplified; (f) cloning the resulting coding elements (A) of step (e) into vectors; (g) transforming the vectors obtained in step (f) into host cells and establishing clonal colonies; and (h) culturing said clonal colonies under conditions suitable to express the coding elements. Also, the method relates to an RNA library or a (poly)peptide library obtainable or obtained according to the method of the invention. Moreover, the invention relates to a method for identifying a (poly)peptide epitope recognized by an antibody or a (poly)peptide-binding compound and a method for identifying a (poly)peptide epitope recognized by antibodies in serum. Further, the invention relates to a method for generating protein variants. Finally, the invention relates to a nucleic acid molecule a ...

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Номер записи: 5

26-01-2012 дата публикации

Synthetic siRNA Detection Method

Номер: US20120021420A1

Автор: Eiji Kobayashi, Hiroyuki Mukai, Satoshi Yanagi, Takashi Uemori

Принадлежит: Takara Bio Inc

A method for accurately and easily detecting a synthetic siRNA, for example, a siRNA in which the 3′ end is DNA, and a kit used for the method are provided. The present invention relates to a method for detecting a siRNA in which the 3′ end is DNA, comprising: (a) adding polydeoxyadenosine to the 3′ DNA end of at least one strand of the siRNA to be detected to produce a polydeoxyadenosine-added RNA; (b) annealing a polydeoxythymidine primer having a tag sequence at its 5′ side to the polydeoxyadenosine-added RNA and synthesizing DNA from the primer by a reverse transcription; and (c) detecting the DNA synthesized in (b).

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Номер записи: 6

09-02-2012 дата публикации

Recombinant vector and use in gene therapy

Номер: US20120034693A1

Автор: Hongzhan Xu

Принадлежит: Individual

A recombinant vector for delivering A3G genes into human cells comprising (i) a gene expression block including an A3G gene selected from a wild type A3G gene represented by SEQ ID NO: 1 and a mutant A3G gene and (ii) a group of elements from a modified lentiviral vector including lentiviral regions of packaging signal (ψ, psi), LTRs, RRE, and PBS; wherein said A3G gene is operably linked to the packaging signal (ψ, psi), LTRs, RRE, and PBS.

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Номер записи: 7

01-03-2012 дата публикации

Method for preparing protein, dna, and rna from cell

Номер: US20120053329A1

Автор: Nobuko Yamamoto

Принадлежит: Canon Inc

An object of the present invention is to prepare DNA, RNA, and a protein from one cell and to provide a convenient preparation method with high reproducibility. To prepare a protein, DNA, and RNA from a cell, nuclear and cytoplasmic separation is performed, and a protein, DNA, and RNA are then extracted.

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Номер записи: 8

15-03-2012 дата публикации

Eukaryotic host cell comprising an expression enhancer

Номер: US20120064630A1

Автор: Diethard Mattanovich, Gerhard Stadlmayr, Michael Sauer

Принадлежит: FH CAMPUS WIEN

Invention relates to a eukaryotic host cell comprising a recombinant nucleotide sequence encoding an expression enhancer, which is selected from the group consisting of cLC52, RPL33 and cLC61, and its use in a method of producing a protein of interest (POI).

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Номер записи: 9

22-03-2012 дата публикации

Methods and systems for uniform enrichment of genomic regions

Номер: US20120071357A1

Автор: Christina Middle, Jacob Kitzman, Jeffrey Jeddeloh, Mark D'Ascanzo, Thomas Albert, Todd Richmond

Принадлежит: Individual

The present invention provides methods and compositions for the enrichment of target nucleic acids in a microarray system. In particular, the present invention provides methods and compositions for uniform enrichment of target nucleic acid molecules in a microarray format. The present invention also provides for intentionally non-uniform enrichment among target nucleic acid molecules.

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Номер записи: 10

05-04-2012 дата публикации

Enrichment of Low Molecular Weight DNA

Номер: US20120083597A1

Автор: Jan Lawrence Godoski, JR., Patricia Okamoto, Thomas Scholl

Принадлежит: Esoterix Genetic Laboratories LLC

The present invention provides, among other things, a simple, reproducible, and cost-effective method for enriching fetal or other low molecular weight nucleic acids in a biological sample. In certain embodiments, methods are provided for enriching fetal nucleic acids (e.g., fetal DNAs), typically comprising steps of adding a polymer such as PEG to a heterogeneous biological sample containing fetal DNA and high molecular weight non-fetal DNA such that the PEG precipitates substantially the high molecular weight non-fetal DNA, and purifying the fetal DNA from supernatant, thereby enriching the fetal DNA.

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Номер записи: 11

12-04-2012 дата публикации

Nucleic acid extraction method

Номер: US20120088226A1

Автор: Geun-bae Lim, Ji-Min Kahng, Jin-Hwa Jung, Tae-Hee Kang

Принадлежит: Academy Industry Foundation of POSTECH, Industry Academic Cooperation Foundation of Catholic University of Korea

The present invention relates to a nucleic acid extracting apparatus, and the nucleic acid extracting apparatus can include a pipe-shaped tube having an open outlet at one side thereof, and a hydrogel column that is provided inside the tube and filters impurities excluding an extraction target material.

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Номер записи: 12

17-05-2012 дата публикации

Dhx36 / rhau knockout mice as experimental models of muscular dystrophy

Номер: US20120124682A1

Автор: Janice Lai, Pu Wu, Yoshikuni Nagamine

Принадлежит: Individual

The present invention provides a genetically-modified non-human animal whose somatic and germ cells contain a gene encoding an altered form of an DHX36 gene, the altered DHX36 haviang been targeted to replace a wild-type DHX36 gene into the animal or an ancestor of the animal at an embyonic stage using embryonic stem cells. An ideal use of the genetically-modified non-human animal of the invention is the use as an experimental model for muscular dystrophy, e.g. spinal muscular atrophy, to identify e.g. new treatments for muscular dystrophy and or study its pathogenesis.

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Номер записи: 13

31-05-2012 дата публикации

Method for Concentrating Sample Constituents and Amplifying Nucleic Acids

Номер: US20120135471A1

Автор: Jan Weile, Martina Daub, Peter Rothacher, Susanne Muench

Принадлежит: ROBERT BOSCH GMBH

A method is disclosed for concentrating sample constituents and for multiplying nucleic acids from a biological sample which are containing in the sample constituents. The nucleic acids are amplified on the same filter on which the sample constituents are also separated off.

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Номер записи: 14

31-05-2012 дата публикации

Methods for Phototransfecting Nucleic Acids Into Live Cells

Номер: US20120135493A1

Автор: Chia-Wen Kitty Wu, Fanyi Zeng, Hajime Takano, Jai-Yoon Sul, James Eberwine, Philip G. Haydon

Принадлежит: University of Pennsylvania Penn

The present invention includes methods for transferring a multigenic phenotype to a cell by transfecting, preferably by phototransfection, and locally transfecting a cell or a cellular process with a laser while the cell is bathed in a fluid medium comprising two or more nucleic acids, thereby introducing the nucleic acid into the interior of the cell. Expression of the nucleic acids results in a multigenic phenotype in the tranfected cell.

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Номер записи: 15

31-05-2012 дата публикации

Constructs expressing chimeric receptors and use thereof for the controlled activation of defence response to pathogens in plants

Номер: US20120137392A1

Автор: Alexandre Brutus, Felice Cervone, Francesca Sicilla, Giulia De Lorenzo

Принадлежит: Universita degli Studi di Roma La Sapienza

The present invention relates to a construct able to express in at least one plant tissue, a chimeric receptor, said chimeric receptor being essentially made of the extracellular region, comprising the external juxtamembrane portion, of a first kinase receptor R1; and the transmembrane region and the intracellular region, comprising the internal juxtamembrane portion, of a second kinase receptor R2, wherein R1 and R2 are different and uses thereof.

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Номер записи: 16

07-06-2012 дата публикации

Recombinant Gene Expression

Номер: US20120142049A1

Автор: Andrew S. Belmont

Принадлежит: Individual

Genes are expressed by culturing cells comprising a host chromosome comprising an integrated artificial chromosome comprising recombinant genes, under conditions whereby each recombinant gene is expressed copy number dependently and position independently. Deletions increase expression from recombinant gene(s) inserted into the artificial chromosome.

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Номер записи: 17

14-06-2012 дата публикации

Methods and compositions for amplification of rna sequences

Номер: US20120149068A1

Автор: Nurith Kurn

Принадлежит: Nugen Technologies Inc

The invention provides methods for isothermal amplification of RNA. The methods are particularly suitable for amplifying a plurality of RNA species in a sample. The methods employ a composite primer, a second primer and strand displacement to generate multiple copies of DNA products comprising sequences complementary to an RNA sequence of interest. In another aspect, the methods employ a single primer (which is a composite primer) and strand displacement to generate multiple copies of DNA products comprising sequences complementary to an RNA sequence of interest. In some embodiments, a transcription step is included to generate multiple copies of sense RNA of an RNA sequence of interest. The methods are useful for preparation of nucleic acid libraries and substrates for analysis of gene expression of cells in biological samples. The invention also provides compositions and kits for practicing the amplification methods, as well as methods which use the amplification products.

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Номер записи: 18

14-06-2012 дата публикации

Methods and compositions for seamless cloning of nucleic acid molecules

Номер: US20120149069A1

Автор: Adam N. Harris, Jonathan D. Chesnut, Knut R. Madden, Louis Leong, Miroslav Dudas

Принадлежит: Life Technologies Corp

The present invention is in the fields of biotechnology and molecular biology. More particularly, the present invention relates to cloning or subcloning one or more nucleic acid molecules comprising one or more type IIs restriction enzyme recognition sites. The present invention also embodies cloning such nucleic acid molecules using recombinational cloning methods such as those employing recombination sites and recombination proteins. The present invention also relates to nucleic acid molecules (including RNA and iRNA), as well as proteins, expressed from host cells produced using the methods of the present invention.

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Номер записи: 19

14-06-2012 дата публикации

Targeted genomic rearrangements using site-specific nucleases

Номер: US20120149115A1

Автор: Eun Ji Kim, Hyung Joo Lee, Jin Soo Kim

Принадлежит: SNU R&DB FOUNDATION, Toolgen Inc

The present invention relates to a method for genomic DNA rearrangements, and more particularly, to a method for deletion, duplication, inversion, replacement, or rearrangement of genomic DNA using pairs of site-specific nucleases targeting two or more sites in the genome, a cell in which genomic DNA is deleted, duplicated, inverted, replaced, or rearranged by the same method, and a method for expressing the site-specific nucleases in cells. Further, the present invention relates to a method for inserting synthetic DNA molecules into the genome using site-specific nucleases targeting a pre-determined site in the genome, a cell in which DNA insertion occurs by the same method, and a method for expressing the site-specific nucleases in cells.

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Номер записи: 20

21-06-2012 дата публикации

Method and Device for Purifying Nucleic Acids

Номер: US20120152818A1

Автор: Dirk Block, Joerg Kleiber, Manuela Poignee-Heger, Ralf Zielenski, Ruediger Haenel, Thomas Walter

Принадлежит: Roche Molecular Systems Inc

The invention concerns a method for isolating and purifying nucleic acids from a sample and a device that is suitable therefore.

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Номер записи: 21

12-07-2012 дата публикации

Screening of protein candidates

Номер: US20120178110A1

Автор: Jianbing Zhang, Tomoko Hirama

Принадлежит: NATIONAL RESEARCH COUNCIL OF CANADA

Successful application of an engineered protein as therapeutics or in other industries would require the protein to have good expression level, good biophysical properties and often desired affinity to its target. The present invention provides a method of screening large numbers of protein candidates (PCs) in all three aspects simultaneously. PCs are fused to a protein anchor, which is captured by the target/antigen. The captured PCs are evaluated for their expression levels, biophysical properties and affinities using conventional methods.

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Номер записи: 22

02-08-2012 дата публикации

Methods for Enriching Microparticles or Nucleic Acids Using Binding Molecules

Номер: US20120196285A1

Автор: Jan Godoski, Patricia Okamoto, Thomas Scholl

Принадлежит: Esoterix Genetic Laboratories LLC

Methods for enriching specific microparticles, such as fetal microparticles or disease specific microparticles, in a biological sample are disclosed. In certain embodiments, the methods include combining a biological sample with a molecule that binds specific microparticles, and separating fractions of the biological sample, wherein the fraction that contains the binding molecule is enriched for the specific microparticles. Also disclosed are methods for enriching fetal nucleic acids by enriching fetal microparticles in a fraction of the biological sample and isolating nucleic acids from the enriched fraction. Methods for facilitating prenatal diagnosis of fetal chromosomal abnormalities are disclosed. In certain embodiments, the methods include combining a biological sample with a molecule that binds fetal microparticles, separating fractions of the biological sample, isolating nucleic acids from the fraction enriched for fetal microparticles, and analyzing the nucleic acids for the presence of a mutation.

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Номер записи: 23

02-08-2012 дата публикации

Isolation of nucleic acids

Номер: US20120197009A1

Автор: Matthew John Baker

Принадлежит: Life Technologies Corp

A method for extracting nucleic acids from a biological material such as blood comprises contacting the mixture with a material at a pH such that the material is positively charged and will bind negatively charged nucleic acids and then eluting the nucleic acids at a pH when the said materials possess a neutral or negative charge to release the nucleic acids. The nucleic acids can be removed under mildly alkaline conditions to the maintain integrity of the nucleic acids and to allow retrieval of the nucleic acids in reagents that are immediately compatible with either storage or analytical testing.

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Номер записи: 24

16-08-2012 дата публикации

Linking sequence reads using paired code tags

Номер: US20120208724A1

Автор: Frank J. Steemers, Igor Yu Goryshin, Kevin Gunderson, Natasha Pignatelli, Nicholas Caruccio, Thomas Royce

Принадлежит: Illumina Inc

Artificial transposon sequences having code tags and target nucleic acids containing such sequences. Methods for making artificial transposons and for using their properties to analyze target nucleic acids.

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Номер записи: 25

13-09-2012 дата публикации

NOVEL MULTIPLEX BARCODED PAIRED-END DITAG (mbPED) SEQUENCING APPROACH AND ITS APPLICATION IN FUSION GENE IDENTIFICATION

Номер: US20120231508A1

Автор: Kuo Ping Chiu, Sheng-Chung Chen, Yi Chou

Принадлежит: Academia Sinica

A method of generating a barcoded Paired-End Ditag (bPED) nucleic acid fragment is disclosed. The method comprises: a) performing a first ligation by ligating a half-adaptor with one or two 3′-overhanging ends to a target nucleic acid to obtain a nucleic acid fragment with two ends each attached to one of the half-adaptor, the half adaptor comprising a half-barcode and a restriction enzyme (RE) recognition site; b) performing a second ligation by ligating two of the half-adaptor at the two ends of the nucleic acid fragment to form a circularized nucleic acid construct, wherein the circularized nucleic acid construct comprises a full-size barcoded adaptor; and c) digesting the circularized nucleic acid construct with a RE that cleaves at a defined distance from the RE recognition site, and thereby generating the bPED nucleic acid fragment.

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Номер записи: 26

13-09-2012 дата публикации

Highly Pure Plasmid DNA Preparations

Номер: US20120231537A1

Автор: Li Chen, Nancy Smyth Templeton

Принадлежит: Gradalis Inc

The present disclosure generally relates to highly pure plasmid compositions having low, or undetectable, levels of colanic acid and other contaminants made by a process that comprises purifying plasmid DNA by chromatography, treating the purified plasmid DNA with a polypeptide that digests colanic acid under conditions that digest the colonic acid, and separating the plasmid DNA from the digested colonic acid.

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Номер записи: 27

20-09-2012 дата публикации

Method for the expression of a recombinant protein in a mammalian cell

Номер: US20120237974A1

Автор: Walter Gerhardus De Vries

Принадлежит: AMARNA HOLDING BV

The invention relates to methods for the production of a recombinant protein in a mammalian cell and methods to enhance the production of recombinant proteins in mammalian cells. More in particular, the invention provides a cell for the production of a recombinant protein of interest wherein said cell is permissive to a polyomavirus and wherein said cell comprises the genetic elements A and B wherein A encodes a polyomaviral large T antigen or a functional equivalent thereof and B comprises a gene encoding a protein of interest under the functional control of a polyomaviral origin of replication or a functional equivalent thereof, wherein said cell lacks the capability to express a polyomaviral small T antigen or a functional equivalent thereof as well as the capability to express a polyomavirus capsid protein.

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Номер записи: 28

20-09-2012 дата публикации

Method for screening and quantifying various enzyme activities using a genetic enzyme screening system

Номер: US20120238470A1

Автор: Eugene Rha, Hee Sik Kim, Jae Jun Song, Jong Hyun Choi, Seung Goo Lee, Su Lim Choi

Принадлежит: Korea Research Institute of Bioscience and Biotechnology KRIBB

A method of detecting and quantifying various enzymatic activities using a constructed artificial genetic circuit GESS (genetic enzyme screening system) for sensing phenolic compounds and a method of screening a trace of activities of target enzymes from a metagenome using the artificial genetic circuit, thereby securing target enzyme genes. When the method for screening and quantifying target enzymatic activity is used, useful genes can be screened from various genetic communities, including environmental or metagenomic libraries, at a single cell level in high throughput (million/day). Further, the sensitivity of the genetic circuit to phenol derivatives and the expression thereof can be controlled, and thus the genetic circuit can rapidly sense and quantify various enzymatic activities. Thus, the method can be advantageously used in the protein engineering technology for enzyme modification. Particularly, it can quantitatively investigate enzymatic activity, and thus can be applied to molecular evolution technology.

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Номер записи: 29

27-09-2012 дата публикации

Riboswitch based inducible gene expression platform

Номер: US20120244601A1

Автор: Carolyn R. Bertozzi, Jessica C. Seeliger, Justin P. Gallivan, Shana Topp

Принадлежит: UNIVERSITY OF CALIFORNIA

The present disclosure provides a synthetic translation regulator, as well as gene expression cassettes and gene expression constructs comprising the synthetic translation regulator. The present disclosure further provides genetically modified bacterial host cells comprising a subject synthetic translation regulator; and methods of regulating gene expression in such host cells.

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Номер записи: 30

11-10-2012 дата публикации

Methods, Compositions, and Kits for Making Targeted Nucleic Acid Libraries

Номер: US20120258892A1

Автор: Yan Wang

Принадлежит: Individual

The present invention provides a method and a kit for selecting and enriching target sequences specific for a genomic region of interest or a subset of a transcriptome using a target-capturing sequence library. The target-capturing sequence library comprises random DNA fragments generated from a target sequence template encompassing all the target sequences. The present invention provides an efficient and cost-effective method of target selection for targeted genome resequencing.

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Номер записи: 31

25-10-2012 дата публикации

Compositions and methods related to synchronous selection of homing peptides for multiple tissues by in vivo phage display

Номер: US20120270808A1

Автор: Mikhail Kolonin, Renata Pasqualini, Wadih Arap

Принадлежит: Individual

Embodiments of the invention include methods for selecting in parallel (i.e., synchronously or simultaneously) peptides that target a number of organs, in which each peptide targets distinct tissues or organs. Typically, the methods of the invention provide for peptide selection in a Minimal number of subjects and still provides a selectively binding peptide. In certain aspects, methods of identifying peptides that bind to multiple selected tissues or organs of an organism may comprise the steps of administering a phage display library to a first subject; obtaining a sample of two or more selected tissues; obtaining phage displaying peptides that bind to the samples from the first subject; enriching for peptides by administering phage isolated from the samples of the first subject to a second subject; obtaining a sample of two or more selected tissues from the second subject; and identifying the peptides displayed.

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Номер записи: 32

25-10-2012 дата публикации

High affinity adaptor molecules for redirecting antibody specifity

Номер: US20120271033A1

Автор: Albert Collinson, Anders Vahlne, Gregor Schurmann, Matti Sallberg, Peter Wagner, Robert Kamen

Принадлежит: OPSONIC THERAPEUTICS Inc

Disclosed are methods for identifying high affinity adaptor molecules that bind to both a circulating antibody and a target molecule and redirect the specificity of the circulating antibody to the target molecule. Exemplary high affinity adaptor molecules are also provided.

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Номер записи: 33

01-11-2012 дата публикации

Gene recombination screening methods

Номер: US20120276527A1

Автор: Rick I. Cohen

Принадлежит: Rutgers State University of New Jersey

Methods and compositions for detecting recombination events are disclosed. Methods and compositions for expressing a gene of interest are also disclosed.

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Номер записи: 34

08-11-2012 дата публикации

Chimeric Endonucleases and Uses Thereof

Номер: US20120284877A1

Автор: Andrea Hlubek, Christian Biesgen, Hans Wolfgang HÖFFKEN

Принадлежит: BASF Plant Science Co GmbH

The invention relates to chimeric endonucleases, comprising a endonuclease and a heterologous DNA binding domain comprising one or more Zn 2 C 6 zinc fmgers, as well as methods of targeted integration, targeted deletion or targeted mutation of polynucleotides using chimeric endonucleases.

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Номер записи: 35

15-11-2012 дата публикации

Probe reagent for measurement of proteolytic activity

Номер: US20120288883A1

Автор: Atsushi Miyawaki, Masahiko Hirano

Принадлежит: JAPAN SCIENCE AND TECHNOLOGY AGENCY, RIKEN Institute of Physical and Chemical Research

This invention relates to a probe reagent comprising, in order from the N-terminus to the C-terminus, the amino acid sequences of a fluorescent protein I, a peptide capable of terminating protein degradation (i.e., a degradation-terminating peptide), a spacer peptide, a fluorescent protein II, and a protein to be degraded, wherein the protein to be degraded is a protein degraded by the ubiquitin-proteasome system, and the probe reagent is degraded from the C-terminus, but that the degradation of the probe reagent is terminated at the degradation-terminating peptide, a nucleic acid encoding the probe reagent, and use of the probe reagent or the nucleic acid.

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Номер записи: 36

15-11-2012 дата публикации

Nucleic acid elution

Номер: US20120289690A1

Автор: Andrew Francis Page, Breck Olland Parker

Принадлежит: GE Healthcare Bio Sciences Corp

This invention relates to the storage on a solid matrix of genetic material, in particular DNA that has been purified prior to the application to the solid matrix. More specifically, the invention relates to a solid matrix for the storage of purified DNA, which matrix has been treated with a solution comprising plant polysaccharide inulin. One advantage of the invention is that an increased amount of DNA can be stored in the solid matrix of the present invention.”

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Номер записи: 37

22-11-2012 дата публикации

Method for isolating small rna

Номер: US20120295328A1

Автор: Ralf Wyrich, Thorsten Voss

Принадлежит: QIAGEN GmbH

A method for isolating small RNA from a sample is provided, the method comprising binding the RNA to silica particles by contacting the sample with a) at least one alcohol, b) at least one chaotropic salt comprising a chaotropic anion selected from the group consisting of trichloroacetate, perchlorate and trifluoroacetate and c) silica particles and separating the bound RNA from the rest of the sample. The present invention also provides compositions and kits to efficiently isolate small RNA molecules from samples, in particular biological samples such as blood, blood products tissue and body fluids.

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Номер записи: 38

29-11-2012 дата публикации

Focused libraries of genetic packages

Номер: US20120302463A1

Автор: Robert Charles Ladner

Принадлежит: Dyax Corp

Focused libraries of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of antibody peptides, polypeptides or proteins and collectively display, display and express, or comprise at least a portion of the focused diversity of the family. The libraries have length and sequence diversities that mimic that found in native human antibodies.

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Номер записи: 39

06-12-2012 дата публикации

Methods and compositions for nucleic acid sample preparation

Номер: US20120309650A1

Автор: Cheryl Heiner, Keith Bjornson, Kevin Travers, Pranav Patel

Принадлежит: Pacific Biosciences of California Inc

Provided are methods and compositions for the production of linear single-stranded nucleic acids, which can be used as templates in high-throughput sequencing systems. Also provided are methods and compositions for the production of closed single-stranded nucleic acid loops, which can be used as templates in high-throughput sequencing systems.

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Номер записи: 40

20-12-2012 дата публикации

Novel Ecdysone Receptor-Based Inducible Gene Expression System

Номер: US20120322148A1

Автор: Dean Ervin Cress, Marianna Zinovjevna Kapitskaya, Subba Reddy Palli

Принадлежит: Intrexon Corp

This invention relates to the field of biotechnology or genetic engineering. Specifically, this invention relates to the field of gene expression. More specifically, this invention relates to a novel inducible gene expression system and methods of modulating gene expression in a host cell for applications such as gene therapy, large scale production of proteins and antibodies, cell-based high throughput screening assays, functional genomics and regulation of traits in transgenic plants and animals.

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Номер записи: 41

03-01-2013 дата публикации

Two-way, portable riboswitch mediated gene expression control device

Номер: US20130004980A1

Автор: Jian-Dong Huang, Ye Jin

Принадлежит: University of Hong Kong HKU

A regulatable gene expression construct comprising a nucleic acid molecule comprising a two-way riboswitch operably linked to a target sequence. Also provided is a library screening strategy for efficient creation of target-specific riboswitches. A theophylline-repressible and IPTG-inducible riboswitch device achieves portable control of gene expression control in a ‘two-way’ manner. The default state of target genes is ON; the targets are switched off by adding theophylline, and switched back to the ON-state by adding IPTG without changing growth medium. The riboswitch device regulates gene expression in a portable, adjustable, and two-way manner with a variety of scientific and biotechnological applications.

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Номер записи: 42

03-01-2013 дата публикации

Nucleic acid encoding reactions

Номер: US20130005585A1

Автор: Andrew May, Brian Fowler, Fiona Kaper, Megan Anderson, Peilin Chen, Robert C. Jones, Ronald Lebofsky

Принадлежит: Fluidigm Corp

Described herein are methods useful for incorporating one or more adaptors and/or nucleotide tag(s) and/or barcode nucleotide sequence(s) one, or typically more, target nucleotide sequences. In particular embodiments, nucleic acid fragments having adaptors, e.g., suitable for use in high-throughput DNA sequencing are generated. In other embodiments, information about a reaction mixture is encoded into a reaction product. Also described herein are methods and kits useful for amplifying one or more target nucleic acids in preparation for applications such as bidirectional nucleic acid sequencing. In particular embodiments, methods of the invention entail additionally carrying out bidirectional DNA sequencing. Also described herein are methods for encoding and detecting and/or quantifying alleles by primer extension.

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Номер записи: 43

17-01-2013 дата публикации

Methods and compositions for enhanced expression and secretion of proteins

Номер: US20130017574A1

Автор: Xiaowu Liu, Zhuying Wang

Принадлежит: Nanjing Jinsirui Science and Technology Biology Corp

Optimized signal peptide coding sequences for enhanced expression and secretion of protein from a cell and related compositions and methods are described. The optimized signal peptide coding sequence encodes an mRNA that contains at least one hairpin structure immediately downstream of the initiation codon. Methods for obtaining the optimized signal peptide coding sequences and methods for enhanced expression and secretion of proteins using the optimized signal peptide coding sequences are also described.

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Номер записи: 44

24-01-2013 дата публикации

Nucleic-acid extraction method and nucleic-acid extraction cartridge

Номер: US20130020201A1

Автор: Kazumine Ito, Michihiro Ohnishi, Naohisa Sakamoto, Tasuku Yotoriyama, Tomohiko Nakamura, Tomoteru Abe

Принадлежит: Sony Corp

The present disclosure provides a nucleic-acid extraction method for carrying out the following procedures in the same cell, the procedures including: performing an ultrasonic process on a sample containing a nucleic acid; adsorbing substances contained in the sample by making use of an adsorption carrier; and condensing the nucleic acid by damming the nucleic acid moving in an electrophoresis phenomenon. In accordance with the nucleic-acid extraction method, it is possible to carry out an ultrasonic process, an adsorption process and an electrophoresis process in the same cell. Thus, it is possible to eliminate an operation to transfer the sample from one cell to another one for each of the processes.

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Номер записи: 45

24-01-2013 дата публикации

Methods and compositions for targeted mutagenesis in bacteria

Номер: US20130023053A1

Автор: John C. March, Matthew S. Russell

Принадлежит: CORNELL UNIVERSITY

This disclosure provides methods and compositions for targeted mutagenesis of specific genes in a bacterial strain. By inducibly over-expressing error-prone polymerases such as Pol IV or Pol V in conjunction with nickase in a bacterial strain, and housing the targeted gene(s) on an episome or plasmid which contains one or more nickase recognition sequences, the targeted gene(s) can be selectively mutated at rates significantly greater than genes contained on the chromosome. The methods disclosed herein are useful for engineering desirable bacterial phenotypes and novel strains, including for example strains useful for treating or degrading waste and/or environmental contaminants, for optimizing bioprocesses, and for converting low-value feed-stock into value-added products.

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Номер записи: 46

24-01-2013 дата публикации

Method for selective isolation and purification of nucleic acids

Номер: US20130023656A1

Автор: Jörg HUCKLENBROICH, Mario Scherer

Принадлежит: QIAGEN GmbH

The present invention relates to a gentle method for isolating and purifying nucleic acids from a biological cell comprising sample comprising at least DNA, RNA, and proteins, comprising at least the steps of 1. mixing the sample with a lysis buffer, 2. incubating the sample at a temperature within a range between 45° C. and 59° C. to obtain DNA as well as RNA or within a range between 60° C. and 70° C., to obtain DNA essentially free of RNA and 3. separating the nucleic acids from any contaminants.

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Номер записи: 47

31-01-2013 дата публикации

Glycine riboswitches, methods for their use, and compositions for use with glycine riboswitches

Номер: US20130029342A1

Автор: Jeffrey Barrick, Maumita Mandal, Ronald R. Breaker

Принадлежит: YALE UNIVERSITY

Riboswitches are structural elements in mRNA that change state when bound by a trigger molecule, and are thus able to regulate gene expression. They can be dissected into two separate domains: one that selectively binds the target (aptamer domain) and another that influences genetic control (expression platform domain). Bacterial glycine riboswitches consist of two tandem aptamer domains which cooperatively bind glycine to regulate the expression of downstream genes. These natural switches are targets for antibiotics and other small molecule therapies. Modified versions of these natural riboswitches can be employed as designer genetic switches that are controlled by specific effector compounds. Disclosed are isolated and recombinant riboswitches, and compositions and methods for selecting and identifying compounds that can activate, inactivate, or block a riboswitch.

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Номер записи: 48

31-01-2013 дата публикации

Method for isolating and purifying nucleic acids

Номер: US20130030163A1

Автор: Jörg HUCKLENBROICH, Mario Scherer, Markus Müller, Roland Fabis

Принадлежит: QIAGEN GmbH

The present invention relates to a method for isolating and purifying nucleic acids, preferably comprising genomic DNA, from biological samples comprising the steps of lysing the sample using a lysis buffer comprising a source of anionic surfactant ions, optionally disintegrating the RNA present in the lysate, precipitating the surfactant ions from the lysate, and separating the nucleic acids from the precipitate and further contaminants by size-exclusion chromatography. The invention furthermore relates to a lysis buffer, a method of lysing cells and a kit for the isolation and purification of nucleic acids.

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Номер записи: 49

07-02-2013 дата публикации

Biological circuit chemotactic converters

Номер: US20130034907A1

Автор: James J. Collins, Kuan-Ta Lu Timothy

Принадлежит: Boston University, Mfassachuetts Inst of Technology

Described herein are novel biological circuit chemotactic converter that utilize modular components, such as genetic toggle switches and single invertase memory modules (SIMMs), for detecting and converting external inputs, such as chemoattractants, into outputs that allow for autonomous chemotaxis in cellular systems. Flexibility in these biological circuit chemotactic converter is provided by combining individual modular components, i.e., SIMMs and genetic toggle switches, together. These biological converter switches can be combined in a variety of network topologies to create network systems that regulate chemotactic responses based on the combination and nature of input signals received.

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Номер записи: 50

28-02-2013 дата публикации

Fragment switch: a reverse genetic approach

Номер: US20130053552A1

Автор: Mohan Balasubramanian, Xie Tang

Принадлежит: Temasek Life Sciences Laboratory Ltd

The present invention relates to the field of reverse genetics. More particularly, the present invention relates to a novel reverse genetic approach termed “fragment switch” which is used to generate an allelic series in genes of interest which are useful for functional analysis.

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Номер записи: 51

07-03-2013 дата публикации

Compositions and methods for use in recombinational cloning of nucelic acids

Номер: US20130059342A1

Автор: David Cheo, Gary F. Temple, James L. Hartley, Michael A. Brasch

Принадлежит: Life Technologies Corp

The present invention relates to compositions and methods for use in recombinational cloning of nucleic acid molecules. In particular, the invention relates to nucleic acid molecules encoding one or more recombination sites or portions thereof, to nucleic acid molecules comprising one or more of these recombination site nucleotide sequences and optionally comprising one or more additional physical or functional nucleotide sequences. The invention also relates to vectors comprising nucleic acid molecules of the invention, to host cells comprising vectors or nucleic acid molecules of the invention, to methods of producing polypeptides using nucleic acid molecules of the invention, and to polypeptides encoded by these nucleic acid molecules or produced by methods of the invention. The invention also relates to the use of these compositions in methods for recombinational cloning of nucleic acids, in vitro and in vivo, to provide chimeric DNA molecules that have particular characteristics and/or DNA segments.

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Номер записи: 52

14-03-2013 дата публикации

ENGINEERED RED-SHIFTED CHANNELRHODOPSIN VARIANTS

Номер: US20130066402A1

Автор: Lin John Yu-luen, Tsien Roger Y.

Принадлежит: The Regents of the University of California

The invention provides engineered red-shifted channelrhodopsin variants. In some embodiments, the channelrhodopsin variants are characterized by improved membrane trafficking, expression, and/or unique spectral and kinetic properties. 1Volvox carteriVolvox carteri. A polypeptide comprising a channelrhodopsin-1 (ChR1) domain , a channelrhodopsin-1 (VChR1) domain and a channelrhodopsin-2 (VChR2) domain.2. The polypeptide of claim 1 , wherein the polypeptide has the structure:{'sup': 1', '2', '3', '4, 'X-X-X-X'}{'sup': '1', 'wherein Xis a ChR1 domain,'}{'sup': '2', 'Xis a first VChR1 domain,'}{'sup': '3', 'Xis a VChR2 domain and'}{'sup': '4', 'Xis a second VChR1 domain.'}3. The polypeptide of claim 1 , having a sequence according to SEQ ID NO: 1.4. The polypeptide of claim 2 , wherein one or more of the domains X claim 2 , X claim 2 , X claim 2 , and Xcomprises 1 claim 2 , 2 claim 2 , 3 claim 2 , 4 claim 2 , or 5 amino acid substitutions relative to a corresponding wild-type domain.5. The polypeptide of claim 4 , wherein the amino acid substitution is at one or more positions selected from 163 claim 4 , 171 claim 4 , 174 and 266.6. The polypeptide of claim 4 , comprising one or more amino acid substitutions selected from Glu163Thr claim 4 , Leu171Ile claim 4 , Leu171Val claim 4 , His174Arg claim 4 , and Phe266Tyr.7. The polypeptide of further comprising a fluorescent polypeptide.8. A polynucleotide encoding the polypeptide of .9. The polynucleotide of claim 8 , having a sequence according to SEQ ID NO: 2.10. A method of depolarizing a cell comprising:{'claim-ref': {'@idref': 'CLM-00001', 'claim 1'}, '(a) expressing the polypeptide of within the cell; and'}(b) exposing the cell to light.11. (canceled)12. (canceled)13. A method of restoring sensitivity to light in an ocular cell comprising expressing the polypeptide of in the ocular cell.14. The polypeptide of claim 1 , comprising an amino acid sequence that is at least 85% identical to SEQ ID NO:1.15. The polypeptide of ...

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Номер записи: 53

21-03-2013 дата публикации

METHODS FOR PRODUCING ANTIBODY-PRODUCING CELLS THAT PRODUCE DESIRED POLYPEPTIDES

Номер: US20130071881A1

Автор: Fujii Shinobu, Kanayama Naoki, Ohmori Hitoshi

Принадлежит: Immuno Tec Laboratory Co. Ltd

An objective of the present invention is to provide methods for introducing DNAs encoding a desired amino acid sequence into a region comprising a DNA encoding an antibody variable region of antibody-producing cells. The present inventors developed methods for efficiently introducing DNAs encoding a desired amino acid sequence into the antibody variable region gene locus of DT40-SW, which is a mutant line of the DT40 chicken B cell line which has the ability to spontaneously introduce mutations. This allows mutagenesis of introduced DNAs to modify the polypeptides to have superior functions. In particular, the present inventors revealed the nucleotide sequence of the antibody H chain variable region gene locus of the DT40 cell line. Based on this finding, the present inventors successfully constructed targeting vectors that allow efficient substitution of the antibody H chain variable region gene locus of the DT40 cell line with a gene encoding a desired polypeptide. 1. A method of homologously recombining a DNA construct and a region comprising a DNA encoding an antibody variable region of an antibody-producing cell , which comprises the step of introducing into the antibody-producing cell a targeting vector comprising a DNA construct that comprises:(1) a promoter DNA that functions in the cell;(2) a DNA that encodes a desired amino acid sequence; and(3) a DNA that inhibits the production of a polypeptide comprising the desired amino acid sequence, and can be removed from the DNA construct.2. The method of claim 1 , wherein the DNA of (3) inhibits the production of a polypeptide comprising the desired amino acid sequence claim 1 , and is located between two site-specific recombinase recognition sequences oriented in the same direction claim 1 , and wherein the DNA of (3) comprises a promoter DNA and a marker gene that function in the cell claim 1 , and the DNA of (3) can be removed from the DNA construct by a site-specific recombinase.3. A method for selecting a ...

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Номер записи: 54

21-03-2013 дата публикации

GLUCOSE VALVE AND OTHER METABOLITE VALVES

Номер: US20130071894A1

Автор: Moon Tae Seok, Prather Kristala Lanett Jones, Solomon Kevin

Принадлежит: Massachusetts Institute of Technology

Aspects of the invention relate to the design and construction of Metabolite Valves, such as Glucose Valves, that can be used to divert metabolites from endogenous pathways toward alternative pathways in a cell. 1. A method for redirecting glycolytic flux in a cell , the method comprising reducing expression of the phosphoenolpyruvate (PEP)-dependent glucose phosphotransferase system (PTS) in the cell and inhibiting phosphorylation of glucose by glucokinase within the cell.2. The method of claim 1 , wherein the cell has increased expression of galactose permease (galP) and/or glucose facilitator protein (glf).3. The method of claim 1 , wherein inhibiting phosphorylation of glucose by glucokinase within the cell comprises reducing expression of glucokinase in the cell.4. The method of claim 3 , wherein reducing expression of glucokinase in the cell comprises recombinantly expressing in the cell an antisense RNA transcript that targets glucokinase.513.-. (canceled)14. The method of claim 1 , wherein expression of glucose-6-phosphate isomerase in the cell is reduced.15. The method of claim 14 , wherein reduced expression of glucose-6-phosphate isomerase in the cell comprises recombinantly expressing in the cell an antisense RNA transcript that targets glucose-6-phosphate isomerase.16. The method of claim 3 , wherein the promoter of glucokinase is replaced by a repressible promoter.17. The method of claim 16 , wherein the cell recombinantly expresses an inducible repressor protein that represses glucokinase expression.18. The method of claim 1 , wherein the cell is a microbial cell.1922.-. (canceled)23. The method of claim 1 , wherein the method is a method of producing gluconate and the method further comprises culturing the cell and optionally recovering gluconate from the cell and/or cell culture claim 1 , or wherein the method is a method of producing glucaric acid and the method further comprises culturing the cell and optionally recovering glucaric acid from the ...

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Номер записи: 55

21-03-2013 дата публикации

ANTIBODIES SPECIFIC FOR DKK-1 AND THEIR USES

Номер: US20130071921A1

Автор: Li Mei, PARALKAR Vishwas Madhav, Pons Jaume, STONE Donna Marie

Принадлежит:

The present invention provides antibodies and fragments thereof that bind to Dkk-1 and, in particular, to humanized antibodies and fragments thereof that bind to Dkk-1 and, even more particularly to fully humanized antibodies and immunologically functional fragments that bind to Dkk-1. Also provided are antibodies and fragments thereof which compete with the binding of an anti-mouse Dkk-1 monoclonal antibody for binding to Dkk-1 cells. Also provided are nucleic acids encoding anti-Dkk-1 antibodies or fragments thereof, as well as expression vectors and host cells incorporating these nucleic acids for the recombinant expression of anti-Dkk-1 antibodies and fragments thereof. Also provided are methods of preparing the antibodies and fragments thereof of the invention. Also provided are bone anabolic agents. Pharmaceutical compositions comprising the antibodies or fragments thereof of the invention are also provided. Further provided are methods of treating diseases, conditions and disorders, such as bone disorders, which result in a loss of bone. Methods of treating or preventing a loss of bone mass, methods of inducing increased bone mass, and methods of inducing Wnt activity are also provided. 1. A recombinant nucleic acid comprising a sequence that encodes (a) a heavy chain variable region (VH) complementary determining region one (CDR1) comprising the amino acid sequence shown in SEQ ID NO: 30 , 49 or 50 , a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 32 or 51 , and a VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 34 or 52; and (b) a light chain variable region (VL) CDR1 comprising the amino acid sequence shown in SEQ ID NO: 22 , a VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 24 , and a VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 26.2. The recombinant nucleic acid of claim 1 , comprising a sequence that encodes: a VH comprising the amino acid sequence shown in SEQ ID NO: 28 claim 1 , a VL ...

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Номер записи: 56

21-03-2013 дата публикации

Anti-C5 Alpha Antibodies

Номер: US20130071922A1

Автор: Marzari Roberto, TEDESCO Francesco

Принадлежит: Adienne SRL

The present invention refers to recombinant antibodies of human origin specific for the C5 component of the activated complement and characterised by the ability to inhibit the conversion of the C5 alpha chain to C5a and C5b. Moreover the present invention refers to the nucleotide sequences coding for such antibodies and to the therapeutic use of both polypeptide and nucleotide sequences, in particular for the therapy of diseases involving tissue damage deriving from uncontrolled activation of the complement system. 1. An isolated nucleotide sequence encoding for a recombinant human antibody having specificity for a C5 alpha chain of a C5 component of the complement system characterized in that it recognizes a region corresponding to sequence 727-744 (SEQ ID NO: 15) of the C5 component of human complement or a region having at least 80% homology thereto , wherein said antibody inhibits the conversion of the C5 alpha chain to C5a and C5b , wherein a light chain of the antibody is a lambda chain or a kappa chain , and a variable region of a heavy chain is the VH3 region.2. The nucleotide sequence according to claim 1 , wherein said antibody further comprises a peptide tag positioned at the C-terminus of said antibody claim 1 , wherein said tag does not alter the binding specificity to the C5 of said antibody.3. The nucleotide sequence according to claim 1 , wherein said nucleotide sequence is effective in treating a disease selected from the group consisting of a chronic inflammatory disease and an acute inflammatory disease.4. The nucleotide sequence according to claim 3 , wherein said acute inflammatory disease is Multiple Organ Failure or myocardial infarction.5. The nucleotide sequence according to claim 3 , wherein said chronic inflammatory disease is selected from the group consisting of: rheumatoid arthritis claim 3 , glomerulonephritis claim 3 , multiple sclerosis claim 3 , demyelinating peripheral neuropathies claim 3 , and atherosclerosis.6. The nucleotide ...

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Номер записи: 57

21-03-2013 дата публикации

Scalable Manufacturing Platform for Viral Vector Purification and Viral Vectors So Purified for Use in Gene Therapy

Номер: US20130072548A1

Автор: Hauck Bernd, High Katherine, QU Guang, Wright John Fraser

Принадлежит:

Methods for preparing highly purified AAV vector formulations are provided. The highly pure AAV formulations described herein are superior for clinical use. 1. A method for purifying bona fide AAV vector particles comprising a transgene encoding a therapeutic protein or fragment thereof from an AAV preparation comprising AAV vector particles , empty capsids and host cell impurities , thereby providing an AAV product substantially free of AAV empty capsids , said method comprising:a) harvesting cells comprising recombinant AAV;b) concentrating said cells via Tangential Flow Filtrationc) lysing said cells by microfluidization to form a lysate;d) filtering, thereby clarifying the lysate of step c);e) purifying AAV particles by Ion Exchange Column Chromatography and optionally concentrating column eluate by Tangential Flow Filtration;f) mixing said eluate with cesium chloride and subjecting said mixture to centrifugation, thereby forming a gradient;g) collecting viral particles separated in step f) and subjecting the same buffer exchange by Tangential Flow Filtration;h) formulating purified AAV particles with surfactant to provide an AAV particle formulation;i) filtering said formulation to remove any remaining impurities, wherein said bona fide AAV vector particles are present in said AAV product in an amount of at least 95%.2. The method of claim 1 , wherein said AAV vector particles are present at a concentration of 100 mg/mL.3. The method of claim 1 , wherein said AAV particles of step i) are present at a concentration of 10particles per mL.4. The method of claim 1 , wherein said AAV particles of step i) are present at a concentration of 10particles per mL.5. The method of claim 1 , wherein said AAV particles of step i) are present at a concentration of 10particles per mL.6. The method of claim 1 , wherein said AAV vector particles are derived from an AAV selected from the group consisting of AAV1 claim 1 , AAV2 claim 1 , AAV5 claim 1 , AAV6 claim 1 , AAV8 and AAV9. ...

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Номер записи: 58

28-03-2013 дата публикации

VECTORS EXPRESSING HIV ANTIGENS AND GM-CSF AND RELATED METHODS OF GENERATING AN IMMUNE RESPONSE

Номер: US20130078276A1

Автор: Amara Rama R., Hellerstein Michael, Lai Lilin, Robinson Harriet L.

Принадлежит:

The disclosure provides vectors encoding one or more HIV antigens and GM-CSF. Also provided are methods of inducing an immune response in a subject, methods of treating a subject having HIV, and methods of manufacturing a medicament for inducing an immune response that require the use of these vectors and vaccine inserts. 1. A vector comprising:a prokaryotic origin of replication;a promoter sequence;a eukaryotic transcription cassette comprising a vaccine insert encoding one or more immunogens and GM-CSF;a polyadenylation sequence; and aa transcription termination sequence.2. The vector of claim 1 , wherein the vaccine insert comprises a sequence that encodes one or more immunogens selected from the group consisting of: gag claim 1 , gp120 claim 1 , pol claim 1 , env claim 1 , Tat claim 1 , Rev claim 1 , Vpu claim 1 , Nef claim 1 , Vif claim 1 , and Vpr.39.-. (canceled)10. The vector of claim 2 , wherein the insert comprises a sequence that encodes gag claim 2 , pol claim 2 , Tat claim 2 , Rev claim 2 , and env.1116.-. (canceled)17. The vector of claim 1 , wherein the encoded GM-CSF is a full-length human GM-CSF.18. The vector of claim 17 , wherein the sequence encoding GM-CSF comprises the sequence of: nucleotides 6633-7068 of SEQ ID NO: 7 claim 17 , nucleotides 6648-7082 of SEQ ID NO: 8 claim 17 , or nucleotides 7336-7770 of SEQ ID NO: 9.1921.-. (canceled)22. The vector of claim 1 , comprising the sequence of GEO-D03 (SEQ ID NO: 7) claim 1 , GEO-D06 (SEQ ID NO: 8) or GEO-D07 (SEQ ID NO: 9).2324.-. (canceled)25. A vaccine insert encoding one or more immunogens and GM-CSF.2643.-. (canceled)44. A method of inducing an immune response in a subject comprising administering to a subject one or more doses of the vector of .45. The method of claim 44 , wherein the vaccine insert comprises a sequence that encodes one or more immunogens selected from the group consisting of: gag claim 44 , gp120 claim 44 , pol claim 44 , env claim 44 , Tat claim 44 , Rev claim 44 , Vpu ...

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Номер записи: 59

28-03-2013 дата публикации

RECOMBINANT MICROORGANISM HAVING AN ABILITY OF USING SUCROSE AS A CARBON SOURCE

Номер: US20130078673A1

Автор: CHOI Sol, KIM Ji Mahn, Lee Jeong Wook, LEE Sang Yup, PARK Jin Hwan, Song Hyohak

Принадлежит: KOREA ADVANCED INSTITUTE OF SCIENCE AND TECHNOLOGY

The present invention relates to a recombinant microorganism capable of metabolizing sucrose, and more particularly to a recombinant microorganism capable of metabolizing sucrose in which a gene encoding sucrose phosphotransferase and/or a gene encoding sucrose-6-phosphate hydrolase is introduced or to a recombinant microorganism capable of metabolizing sucrose in which a gene encoding β-fructofuranosidase is introduced. According to the present invention, a recombinant microorganism capable of using inexpensive sucrose as a carbon source instead of expensive glucose is provided. In addition, in a process of culturing microorganisms which have been incapable of using sucrose as a carbon source, sucrose can substitute for other carbon sources including glucose. 122.-. (canceled)23. A recombinant vector containing a gene (ptsG) encoding a sucrose phosphotransferase and a sacC gene encoding sucrose-6-phosphate hydrolase , wherein the ptsG gene is represented by a base sequence of SEQ ID NO:2.24. The recombinant vector according to claim 23 , wherein the sacC gene is represented by a base sequence of SEQ ID NO: 4.25. A recombinant microorganism capable of metabolizing sucrose in which the recombinant vector of is introduced into a host cell selected form the group consisting of bacteria claim 23 , yeast and fungi.26. A recombinant microorganism capable of metabolizing sucrose in which a gene (ptsG) encoding a sucrose phosphotransferase and a gene (sacC) encoding sucrose-6-phosphate hydrolase is introduced into a chromosomal DNA of a host cell selected form the group consisting of bacteria claim 23 , yeast and fungi claim 23 , wherein the ptsG gene is represented by a base sequence of SEQ ID NO:2.27. The recombinant microorganism capable of metabolizing sucrose according to claim 26 , wherein the sacC gene is represented by a base sequence of SEQ ID NO: 4.28. A recombinant microorganism capable of metabolizing sucrose in which a gene encoding a β-fructofuranosidase ...

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Номер записи: 60

04-04-2013 дата публикации

Novel Therapeutic RNA Interference Technology Targeted to the PDX-1 Oncogene in PDX-1 Expressing Neuroendocrine Tumors

Номер: US20130084331A1

Автор: Brunicardi F. Charles, Nemunaitis John J., Rao Donald

Принадлежит:

A bifunctional shRNA-based composition and methods for knocking down the expression of the PDX-1 oncogene in target cells is described herein. The invention also provides methods to deliver the shRNA-containing expression vectors to target tissues overexpressing the PDX-1 oncogene. 1. An expression vector comprising:a promoter; anda nucleic acid insert operably linked to the promoter, wherein the insert encodes one or more short hairpin RNAs (shRNA) capable of hybridizing to a region of an mRNA transcript that encodes a PDX-1 oncogene and that inhibits the PDX-1 oncogene expression via RNA interference.2. The expression vector of claim 1 , wherein the shRNA incorporates one or more siRNA (cleavage-dependent) and miRNA (cleavage-independent) motifs.3. The expression vector of claim 1 , wherein the shRNA is both the cleavage-dependent and cleavage-independent inhibitor of the PDX-1 oncogene expression.4. The expression vector of claim 1 , wherein the shRNA is further defined as a bifunctional shRNA.5. The expression vector of claim 1 , wherein the one or more shRNA comprise a sequence selected from the group consisting of SEQ. ID NO: 3 claim 1 , SEQ. ID NO: 4 claim 1 , SEQ. ID NO: 5 claim 1 , SEQ. ID NO: 6 claim 1 , SEQ. ID NO: 7 claim 1 , SEQ. ID NO: 8 claim 1 , and combinations or modifications thereof.6. The expression vector of claim 1 , wherein the region targeted is the 3′ UTR region sequence of the PDX-1 oncogene transcript.7. A therapeutic delivery system comprising:a therapeutic agent carrier; andan expression vector comprising a promoter and a nucleic acid insert operably linked to the promoter encodes one or more short hairpin RNA (shRNA) that hybridizes to a region of an mRNA transcript that encodes a PDX-1 oncogene and inhibits the PDX-1 oncogene expression via RNA interference.8. The delivery system of claim 7 , wherein the therapeutic agent carrier is a compacted DNA nanoparticle.9. The delivery system of claim 8 , wherein the DNA nanoparticle is ...

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Номер записи: 61

04-04-2013 дата публикации

DECREASING LACTATE LEVEL AND INCREASING POLYPEPTIDE PRODUCTION BY DOWNREGULATING THE EXPRESSION OF LACTATE DEHYDROGENASE AND PYRUVATE DEHYDROGENASE KINASE

Номер: US20130084605A1

Автор: Ng Chi Kin Domingos, SHEN Amy, SNEDECOR Bradley Richard, Zhou Meixia

Принадлежит: Genentech, Inc.

The present invention provides methods and compositions for reducing lactate production and increasing polypeptide production in cultured cells. In one aspect, the invention provides a method comprising culturing cells expressing a) a small interfering RNA (siRNA) specific for a lactate dehydrogenase (LDH) and b) an siRNA specific for a pyruvate dehydrogenase kinase (PDHK). In another aspect, the invention provides cultured cells or vectors comprising an siRNA specific for a LDH and an siRNA specific for a PDHK. 1. A method for reducing lactate production in cultured cells , the method comprising culturing cells comprising a first heterologous nucleic acid sequence encoding a small interfering RNA (siRNA) specific for a lactate dehydrogenase (LDH) and a second heterologous nucleic acid sequence encoding an siRNA specific for a pyruvate dehydrogenase kinase (PDHK) , wherein the first heterologous nucleic acid sequence is operably linked to a first promoter , and wherein the second heterologous nucleic acid sequence is operably linked to a second promoter.2. The method of claim 1 , wherein the LDH is LDHa.3. The method of claim 1 , wherein the cultured cells further comprise a third heterologous nucleic acid sequence encoding an siRNA specific for a second PDHK and wherein the third heterologous nucleic acid sequence is operably linked to a third promoter.4. The method of claim 3 , wherein the cultured cells further comprise a fourth heterologous nucleic acid sequence encoding an siRNA specific for a third PDHK and wherein the fourth heterologous nucleic acid sequence is operably linked to a fourth promoter.5. The method of claim 4 , wherein cultured cells further comprise a fifth heterologous nucleic acid sequence encoding an siRNA specific for a fourth PDHK and wherein the fifth heterologous nucleic acid sequence is operably linked to a fifth promoter.65. The method of any one of claim 4 , claim 4 , claim 4 , and claim 4 , wherein the PDHK is selected from the group ...

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Номер записи: 62

04-04-2013 дата публикации

SUPERCOILED MINICIRCLE DNA FOR GENE THERAPY APPLICATIONS

Номер: US20130085173A1

Автор: Bakkalbasi Erol, Catanese Daniel James, Fogg Jonathan, Gilbert Brian E., Zechiedrich E. Lynn

Принадлежит: Baylor College of Medicine

The present invention relates to nucleic acid molecule compositions comprising minivectors encoding a nucleic acid sequence and methods of gene therapy and prophylaxis against infection using minivectors encoding a nucleic acid sequence. 1. A nucleic acid molecule composition comprising a minivector , wherein said minivector encodes a nucleic acid sequence that comprises short hairpin RNA (shRNA) against a target on influenza virus.2. The composition of claim 1 , wherein the minivector comprises a chemical moiety claim 1 , a modified oligonucleotide claim 1 , and/or a modified backbone.3. The composition of claim 2 , wherein the chemical moiety is selected from the group consisting of: fluorescein claim 2 , biotin claim 2 , a dye claim 2 , and cholesterol.4. The composition of claim 1 , wherein the target on influenza virus is a conserved influenza packaging signal.5. The composition of claim 4 , wherein the conserved influenza packaging signal is a packaging signal on a gene selected from: PA claim 4 , PB 1 and PB2.6. A method of silencing expression of an influenza gene in a cell claim 4 , comprising contacting said cell with a minivector claim 4 , wherein said minivector encodes a nucleic acid sequence claim 4 , wherein the nucleic acid sequence silences the expression of the gene.7. The method of claim 6 , wherein the nucleic acid sequence comprises short hairpin RNA (shRNA).8. The method of claim 6 , wherein the nucleic acid sequence comprises DNA that can be bound by a component selected from the group consisting of: a protein; a different DNA sequence; an RNA sequence; and a cell membrane.9. The method of claim 6 , wherein the nucleic acid sequence comprises a gene.10. The method of claim 6 , wherein the nucleic acid sequence comprises short hairpin RNA (shRNA) against a conserved influenza packaging signal.11. The method of claim 10 , wherein the conserved influenza packaging signal is a packaging signal on a gene selected from: PA claim 10 , PB1 and PB2.12. ...

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Номер записи: 63

04-04-2013 дата публикации

PHARMACEUTICAL COMPOSITION UTILIZING PANCREATIC BETA CELL PROLIFERATION FACTOR

Номер: US20130086704A1

Автор: Handa Hiroshi, Imai Takeshi

Принадлежит: TOKYO INSTITUTE OF TECHNOLOGY

Disclosed are a pharmaceutical composition, a screening method, and the like which use UDP-glucose glycoprotein glycosyl transferase 1 (UGGT1) or a gene encoding the same. UGGT1 has an extremely high proliferative activity compared to known pancreatic β-cell proliferation factors; thus, UGGT1 can act as a useful therapeutic agent for diabetes without any modification and is also useful as a target substance for the development of a new therapeutic agent for diabetes. 1. A pharmaceutical composition comprising a vector for expressing UGGT1 gene.2. The pharmaceutical composition according to claim 1 , wherein the composition is used for the prevention or treatment of diabetes.3. A method comprising administering the pharmaceutical composition according to or to a non-human animal and proliferating pancreatic β-cells in the non-human animal.4. A transgenic non-human animal claim 1 , wherein UGGT1 gene is introduced thereinto and the gene is expressed.5. A cell transformed with a vector in which a reporter gene is linked downstream of a promoter of UGGT1 gene.6. The cell according to claim 5 , wherein the cell is a pancreatic β-cell.7. A screening method for a preventive agent or a therapeutic agent for diabetes claim 5 , comprising the steps of culturing the cell according to or in the presence of each test substance; and selecting a test substance having increased the expression level of the reporter gene.8. A screening method for a preventive agent or a therapeutic agent for diabetes claim 5 , comprising the steps of mixing each test substance with UGGT1 and selecting test substances interacting with UGGT1; and administering each of the selected test substances to a non-human animal and selecting a test substance proliferating pancreatic β-cells in the non-human animal. The present invention relates to a pharmaceutical composition, a screening method, and the like which use UDP-glucose glycoprotein glycosyl transferase 1 (hereinafter referred to as “UGGT1”) or a gene ...

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Номер записи: 64

18-04-2013 дата публикации

METHOD FOR CONSTRUCTING RECOMBINANT BACTERIUM FOR PRODUCING NON-NATIVE PROTEIN, AND UTILIZATION OF SAME

Номер: US20130095524A1

Автор: Matsumoto Akiko, Mukai Takahito, Sakamoto Kensaku, Yokoyama Shigeyuki

Принадлежит: RIKEN

The present invention provides a novel method of producing a recombinant bacterium for production of a non-natural protein, including: (1) expressing tRNA in a bacterium, which tRNA recognizes UAG codon; (2) expressing an aminoacyl-tRNA synthetase in the bacterium, which aminoacyl-tRNA synthetase acylates the tRNA with a non-natural amino acid or an α-hydroxy acid; (3) (i) introducing a DNA construct into the bacterium, which DNA construct is for expressing, in the absence of a release factor for terminating translation at UAG codon, a function of at least one gene selected from the group consisting of genes each of which loses its function when a gene that codes for the release factor is defective and/or introducing an alteration into said at least one gene in a chromosome of the bacterium, which alteration is for expressing the function of said at least one gene in the absence of the release factor; and (4) causing the gene that codes for the release factor in the bacterium to be defective. 1. A method of producing a recombinant bacterium for production of a non-natural protein , comprising the steps of:(1) expressing tRNA in a bacterium, which tRNA recognizes UAG codon;(2) expressing an aminoacyl-tRNA synthetase in a bacterium, which aminoacyl-tRNA synthetase acylates the tRNA with a non-natural amino acid or an α-hydroxy acid;(3) subjecting a bacterium to a process for expressing, in the absence of a release factor for terminating translation at UAG codon, a function of at least one gene selected from the group consisting of genes each of which loses its function when a gene that codes for the release factor is defective; and(4) causing the gene that codes for the release factor in a bacterium to be defective,the bacteria in the steps (1) through (4) being identical, andthe process being (i) a process of introducing, into the bacterium, a DNA construct for expressing the function of said at least one gene in the absence of the release factor and/or (ii) a ...

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Номер записи: 65

25-04-2013 дата публикации

METHODS FOR REPROGRAMMING SOMATIC CELLS

Номер: US20130102080A1

Автор: Hochedlinger Konrad, Jaenisch Rudolf

Принадлежит: WHITEHEAD INSTITUTE FOR BIOMEDICAL RESEARCH

The invention provides methods for reprogramming somatic cells to generate multipotent or pluripotent cells. Such methods are useful for a variety of purposes, including treating or preventing a medical condition in an individual. The invention further provides methods for identifying an agent that reprograms somatic cells to a less differentiated state. 1. A method of making a somatic cell more susceptible to reprogramming to a less differentiated state comprising increasing expression of Oct4 protein in the somatic cell , wherein increased expression of Oct4 protein makes the cell more susceptible to reprogramming to a less differentiated state.2. The method of claim 1 , wherein increasing expression of Oct4 protein in the somatic cell comprises introducing at least one exogenous nucleic acid into the somatic cell claim 1 , wherein the exogenously introduced nucleic acid increases expression of Oct4 claim 1 , thereby making the cell more susceptible to reprogramming to a less differentiated state.3. The method of claim 2 , wherein the exogenous nucleic acid encodes Oct4 claim 2 , Nanog claim 2 , or Sox2.4. The method of claim 2 , wherein the endogenous nucleic acid encodes Oct4 or Nanog.5. The method of claim 1 , wherein the somatic cell does not comprise a selectable marker integrated into the endogenous locus of the pluripotency gene.6. The method of claim 1 , wherein the somatic cell is a human cell or a mouse cell.7. The method of claim 1 , wherein the somatic cell is an adult stem cell.8. The method of claim 7 , wherein the adult stem cell is a hematopoietic stem cell claim 7 , neural stem cell claim 7 , or mesenchymal stem cell. This application is a continuation of U.S. application Ser. No. 12/703,061, filed Feb. 9, 2010, which is a divisional of U.S. application Ser. No. 10/997,146, filed Nov. 24, 2004 (U.S. Pat. No. 7,682,828), which claims the benefit of U.S. Provisional Application No. 60/525,612, filed Nov. 26, 2003, and U.S. Provisional Application No ...

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Номер записи: 66

02-05-2013 дата публикации

POLYPEPTIDE PRODUCING CELLS

Номер: US20130109058A1

Автор: Endl Josef, KOPETZKI Erhard, Ploettner Oliver, Schwarz Ursula, TIEFENTHALER Georg

Принадлежит: Hoffmann-La Roche Inc.

The current invention describes a nucleic acid comprising in a 5′ to 3′ direction a) a first nucleic acid encoding a heterologous polypeptide without an in frame stop codon, b) a second nucleic acid beginning with a 5′ splice donor site and terminated by a 3′ splice acceptor site comprising an in frame translational stop codon and a polyadenylation signal, and c) a nucleic acid encoding i) at least a fragment of a transmembrane domain, or ii) a signal peptide for a GPI-anchor. 1. A vector comprising in a 5′ to 3′ directiona) a first multiple cloning site, i) said nucleic acid comprises a stop codon and a polyadenylation signal, and', 'ii) said nucleic acid is not constitutively removed during pre-mRNA processing, and, 'b) a nucleic acid beginning with a 5′ splice donor site and terminated by a 3′ splice acceptor site, wherein'}c) a second multiple cloning site.2. A method for the production of a heterologous polypeptide or protein encoded by a nucleic acid , comprising ,a) providing a eukaryotic cell, i) a first nucleic acid encoding an immunoglobulin heavy chain without an in frame stop codon at its 3′ terminus,', 'ii) a second nucleic acid beginning with a 5′ splice donor site and terminated by a 3′ splice acceptor site comprising an in frame translational stop codon and a polyadenylation signal of a human or mouse immunoglobulin α, ε, γ and μ heavy chain, and', 'iii) a third nucleic acid encoding at least 60% of a complete human immunoglobulin transmembrane domain,, 'b) transfecting said eukaryotic cell with a nucleic acid, wherein the nucleic acid comprises in a 5′ to 3′ direction'}wherein said immunoglobulin heavy chain and a marker are expressed from the same nucleic acid, wherein the marker is a plasma-membrane bound form of the expressed immunoglobulin heavy chain,c) culturing said transfected cell under conditions suitable for the expression of said nucleic acid,d) recovering said polypeptide or protein from the culture medium or the cytoplasm of the cells. ...

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Номер записи: 67

02-05-2013 дата публикации

Method for Producing an L-Amino Acid Using a Bacterium of the Enterobacteriaceae Family

Номер: US20130109063A1

Автор: Gusyatiner Mikhail Markovich, Kiryukhin Mikhail Yurievich

Принадлежит: AJINOMOTO CO., INC.

The present invention provides a method for producing L-amino acid using a bacterium of the Enterobacteriaceae family, particularly a bacterium belonging to the genus or , which has been modified to enhance the expression of the bssR gene, which encodes a regulator of biofilm through signal secretion. 1. An L-amino acid-producing bacterium of the Enterobacteriaceae family , wherein said bacterium has been modified to enhance the expression of the bssR gene.2. The bacterium according to claim 1 , wherein said expression is enhanced by modifying an expression control sequence of the bssR gene.3Escherichia.. The bacterium according to claim 1 , wherein said bacterium belongs to genus4Escherichia coli.. The bacterium according to claim 3 , wherein said bacterium is5Pantoea.. The bacterium according to claim 1 , wherein said bacterium belongs to genus6. The L-amino acid-producing bacterium according to claim 1 , wherein said L-amino acid is selected from the group consisting of an aromatic L-amino acid and a non-aromatic L-amino acid.7. The L-amino acid-producing bacterium according to claim 6 , wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine claim 6 , L-tyrosine claim 6 , and L-tryptophan.8. The L-amino acid-producing bacterium according to claim 6 , wherein said non-aromatic L-amino acid is selected from the group consisting of L-threonine claim 6 , L-lysine claim 6 , L-cysteine claim 6 , L-methionine claim 6 , L-leucine claim 6 , L-isoleucine claim 6 , L-valine claim 6 , L-histidine claim 6 , glycine claim 6 , L-serine claim 6 , L-alanine claim 6 , L-asparagine claim 6 , L-aspartic acid claim 6 , L-glutamine claim 6 , L-glutamic acid claim 6 , L-proline claim 6 , L-arginine claim 6 , L-citrulline claim 6 , and L-ornithine.9. The L-amino acid-producing bacterium according to claim 1 , wherein said L-amino acid is L-arginine.10. A method for producing an L-amino acid comprising cultivating the bacterium according to in a ...

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Номер записи: 68

02-05-2013 дата публикации

METHODS FOR REPROGRAMMING SOMATIC CELLS

Номер: US20130109089A1

Автор: Hochedlinger Konrad, Jaenisch Rudolf

Принадлежит: WHITEHEAD INSTITUTE FOR BIOMEDICAL RESEARCH

The invention provides methods for reprogramming somatic cells to generate multipotent or pluripotent cells. Such methods are useful for a variety of purposes, including treating or preventing a medical condition in an individual. The invention further provides methods for identifying an agent that reprograms somatic cells to a less differentiated state. 1. A composition comprising an isolated primary somatic cell that comprises an exogenously introduced nucleic acid , wherein the exogenously introduced nucleic acid increases Oct4 expression in the cell.2. The composition of claim 1 , wherein the isolated primary somatic cell is a mammalian cell.3. The composition of claim 1 , wherein the isolated primary somatic cell is a human cell or a mouse cell.4. The composition of claim 1 , wherein the isolated primary somatic cell is an adult stem cell.5. The composition of claim 4 , wherein the adult stem cell is selected from the group consisting of: a hematopoietic stem cell claim 4 , a neural stem cell claim 4 , and a mesenchymal stem cell.6. The composition of claim 1 , further comprising a candidate agent of interest with respect to its potential to reprogram a somatic cell.7. The composition of claim 6 , wherein the candidate agent of interest is a DNA methylation inhibitor claim 6 , a histone deacetylase inhibitor or PD098059.8. The composition of claim 6 , wherein the candidate agent of interest is a pluripotency protein selected from the group consisting of: Oct4 claim 6 , Nanog claim 6 , and Sox-2.9. The composition of any one of to claim 6 , wherein the isolated primary somatic cell comprises an isolated nucleic acid encoding a Nanog protein or a Sox2 protein.10. A somatic cell reprogramming composition comprising an isolated nucleic acid encoding an Oct4 protein claim 6 , and a candidate agent of interest with respect to its potential to reprogram a somatic cell.11. A somatic cell reprogramming composition comprising an isolated nucleic acid encoding an Oct4 ...

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Номер записи: 69

02-05-2013 дата публикации

METHODS FOR REPROGRAMMING SOMATIC CELLS

Номер: US20130109090A1

Автор: Hochedlinger Konrad, Jaenisch Rudolf

Принадлежит: WHITEHEAD INSTITUTE FOR BIOMEDICAL RESEARCH

The invention provides methods for reprogramming somatic cells to generate multipotent or pluripotent cells. Such methods are useful for a variety of purposes, including treating or preventing a medical condition in an individual. The invention further provides methods for identifying an agent that reprograms somatic cells to a less differentiated state. 1. A reprogrammed somatic cell comprising an amount of Oct4 expression comparable to the amount of Oct4 expression in an embryonic stem cell , wherein the reprogrammed somatic cell is not an embryonic stem cell.2. A somatic cell comprising an amount of Oct4 protein sufficient to reprogram the cell to a less differentiated state.3. The somatic cell of any one of to , wherein the somatic cell is a mammalian cell.4. The somatic cell of any one of to , wherein the somatic cell is a human cell or a mouse cell.5. The somatic cell of any one of to , wherein the cell is an adult stem cell.6. The somatic cell of claim 5 , wherein the adult stem cell is selected from the group consisting of: a hematopoietic stem cell claim 5 , a neural stem cell claim 5 , and a mesenchymal stem cell.7. A somatic cell reprogramming composition comprising an isolated nucleic acid encoding an Oct4 protein operably linked to at least one regulatory sequence.8. A somatic cell reprogramming composition comprising an isolated nucleic acid encoding an Oct4 protein operably linked to at least one regulatory sequence and an isolated nucleic acid encoding a Sox2 protein operably linked to at least one regulatory sequence.9. A somatic cell reprogramming composition comprising an isolated nucleic acid encoding an Oct4 protein operably linked to at least one regulatory sequence and an isolated nucleic acid encoding a Nanog protein operably linked to at least one regulatory sequence.10. The composition of any one of to claim 5 , wherein the somatic cell is a human cell or a mouse cell.11. The composition of any one of to claim 5 , wherein the somatic cell ...

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Номер записи: 70

02-05-2013 дата публикации

HIGH EFFICIENCY, SMALL VOLUME NUCLEIC ACID SYNTHESIS

Номер: US20130109596A1

Автор: PETERSON Todd C., POEHMERER Thomas, TREFZER Axel C.

Принадлежит: LIFE TECHNOLOGIES CORPORATION

The disclosure generally relates to compositions and methods for the production of nucleic acid molecules. In some aspects, the invention allows for the microscale generation of nucleic acid molecules, optionally followed by assembly of these nucleic acid molecules into larger molecules. In some aspects, the invention allows for efficient production of nucleic acid molecules (e.g., large nucleic acid molecules such as genomes). 1. A multiwell plate for non-template directed synthesis of nucleic acid molecules , the plate comprising:(a) a magnetic bead located in each of a plurality of wells of the plate, and(b) an electrochemically generated acid being present in one or more well, wherein the bead is between 1.0 μm and 100 μm in diameter.2. The multiwell plate of claim 1 , wherein the number of wells in the plate is between 10 and 50 claim 1 ,000.3. The multiwell plate of claim 1 , wherein the total volume of each well is between 0.1 μl and 50 μl.4. The multiwell plate of claim 1 , wherein each well is operably connected to a pair of electrodes.5. The multiwell plate of claim 1 , wherein the wells of the plate are connected to microfluidic channels for the introduction and removal of reagents.6. A method for the generation of an assembled nucleic acid molecule claim 1 , the method comprising:(a) synthesizing a plurality of nucleic acid molecules, wherein each nucleic acid molecule is prepared in a well of a plate in an average amount of from about 0.001 nanomoles to about 1,000 nanomoles;(b) combining the nucleic acid molecules generated in (a) to produce a pool;(c) oining some or all of the nucleic acid molecules present in the pool formed in (b) to form a plurality of larger nucleic acid molecules;(d) eliminating nucleic acid molecules which contain sequence errors from the plurality of larger nucleic acid molecules formed in (c) to produce an error corrected nucleic acid molecule pool; and(e) assembling the nucleic acid molecules in the error corrected nucleic ...

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Номер записи: 71

09-05-2013 дата публикации

Expression Vector

Номер: US20130115219A1

Автор: Beauchamp Jeremy, Dreyer Anita, Matile Hugues

Принадлежит: F. Hoffmann-La Roche AG

The present invention provides an expression vector for cell-surface expression of proteins. 1. A nucleic acid expression vector for cell-surface expression of proteins comprising in order a polynucleotide sequence comprising a sequence encoding a secretion signal peptide , a cloning site for inserting a polynucleotide sequence encoding a protein to be expressed and a polynucleotide sequence comprising a sequence encoding a transmembrane domain of glycophorin.2. The nucleic acid vector of claim 1 , wherein the transmembrane domain of glycophorin is the transmembrane domain of glycophorin A.3. The nucleic acid vector of or claim 1 , wherein the transmembrane domain of glycophorin A is the mouse glycophorin A transmembrane domain or Armenian hamster glycophorin A domain.4. The nucleic acid vector of claim 3 , wherein the mouse glycophorin A domain comprises the amino acids disclosed in Seq. Id. No. 1 and the Armenian hamster glycophorin A domain comprises the amino acid sequence disclosed in Seq. Id. No. 12.54. The nucleic acid vector of - claims 1 , wherein the secretion signal peptide is the secretion signal peptide of bee-venom melittin.6. The nucleic acid vector of claim 5 , wherein the secretion signal peptide of bee-venom melittin comprises the amino acid sequence disclosed in Seq. Id. No. 2.76. The nucleic acid vector of - claims 1 , further comprising downstream (3′) of the cloning site for inserting a polynucleotide sequence encoding a protein to be expressed a polynucleoide sequence encoding a FLAG tag comprising the amino acid sequence of Seq. Id. No. 3.87. The nucleic acid vector of - claims 1 , further comprising downstream (3′) of the polynucleotide sequence encoding the transmembrane domain of glycophorin a polynucleotide sequence encoding a His tag claims 1 , preferably a His tag comprising the amino acid sequence disclosed in Seq. Id. No. 4.98. The nucleic acid vector of - claims 1 , wherein the cloning site comprises the restriction enzyme cleavage ...

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Номер записи: 72

09-05-2013 дата публикации

Reversible, parallel and multitask cloning method and kit

Номер: US20130115658A1

Автор: Cedric Szpirer, Michel C. Milinkovitch, Philippe Gabant

Принадлежит: Individual

The present invention is related to integrated method and tools to construct recombinant DNA molecules (to be used as DNA vaccine or gene therapy) without requiring the use of antibiotic(s) resistance gene(s) and without requiring the addition of one or more antibiotic(s) to the culture medium of cells submitted to this recombinant DNA method. The present invention allows to obtain the selection of recombinant host cell(s) transformed by a (exogenous) nucleic acid sequence of interest (extra-chromosomal vector containing the insert) and simultaneously stabilization (stable inheritance) of this (exogenous) nucleic acid sequence of interest into the transformed host cell(s) descendants (maintenance of the nucleic acid sequence of interest in the host cells population).

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Номер записи: 73

09-05-2013 дата публикации

Method for detecting target molecules

Номер: US20130116129A1

Автор: Makoto Miyagishi

Принадлежит: National Institute of Advanced Industrial Science and Technology AIST

The present invention provides a method for detecting a target molecule using the sequence information of a collection (pool) of aptamers capable of specifically binding to a target molecule, comprising the following steps of: (a) bringing a target molecule into contact with a collection of oligonucleotides which comprise multiple nucleic acid aptamers having different randomized sequences; (b) selecting a subcollection of oligonucleotides that bind to the target molecule; (c) examining the sequence of each oligonucleotide of the selected subcollection; (d) extracting sequence information which is characteristic to the oligonucleotides having affinity for the target molecule, from the sequences of the selected oligonucleotides; and (e) identifying the target molecule based on the sequence information.

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Номер записи: 74

09-05-2013 дата публикации

Production of Post-Translationally Hydroxylated Recombinant Proteins in Bacteria

Номер: US20130116412A1

Автор: Barron Annelise E., Ding Sheng, Pinkas Daniel M.

Принадлежит:

Bacterial cells capable of producing recombinant proteins, such as post-translationally hydroxylated recombinant proteins, methods and kits for producing recombinant proteins, such as post-translationally hydroxylated recombinant proteins, and particular post-translationally hydroxylated recombinant collagen molecules produced by the methods and cells disclosed herein are provided by this invention. 1. A bacterial cell capable of expressing recombinant proteins comprising:a) one or more nucleic acids encoding a sugar-1,4-lactone oxidase or a sugar-1,4-lactone dehydrogenase; andb) one or more nucleic acids encoding an ascorbate-dependent biosynthetic enzyme.2. The bacterial cell of claim 1 , wherein the one or more nucleic acids encoding the sugar-1 claim 1 ,4-lactone oxidase or sugar-1 claim 1 ,4-lactone dehydrogenase comprise a first expression vector claim 1 , and the one or more nucleic acids encoding the ascorbate-dependent biosynthetic enzyme comprise a second expression vector.3. The bacterial cell of claim 1 , wherein the nucleic acids encoding the sugar-1 claim 1 ,4-lactone oxidase or sugar-1 claim 1 ,4-lactone dehydrogenase and the ascorbate-dependent biosynthetic enzyme comprise a single expression vector.4. The bacterial cell of claim 1 ,wherein the sugar-1,4-lactone oxidase is D-arabinono-1,4-lactone oxidase, L-gulono-1,4-lactone oxidase, or D-glucono-1,4-lactone oxidase; andwherein the sugar-1,4-lactone dehydrogenase is D-arabinose dehydrogenase, L-gulono-1,4-lactone dehydrogenase, L-gulono-γ-lactone dehydrogenase, D-glucose dehydrogenase, L-galactono-1,4-lactone dehydrogenase, L-galactono-γ-lactone dehydrogenase, L-sorbosone dehydrogenase, or 2-ketogluconate dehydrogenase.5. (canceled)6. The bacterial cell of claim 1 , wherein the ascorbate-dependent biosynthetic enzyme is a hydroxylase claim 1 , wherein the hydroxylase is prolyl-4-hydroxylase claim 1 , prolyl-3-hydroxylase claim 1 , lysyl-5-hydroxylase claim 1 , HIF prolyl hydroxylase claim 1 , ...

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Номер записи: 75

16-05-2013 дата публикации

METHODS, COMPOSITIONS AND KITS FOR ONE-STEP DNA CLONING USING DNA TOPOISOMERASE

Номер: US20130122572A1

Автор: Minshull Jeremy S., Ness Jon E.

Принадлежит: DNA TWOPOINTO, INC.

Provided are methods, compositions, and kits for cloning of DNA using DNA topoisomerase. The methods comprise (I) combining into a mixture (A) a first polynucleotide comprising an origin of replication, a selectable marker, two topoisomerase recognition sequences, and two nicking agent recognition sequences, each of the topoisomerase recognition sequences being within 50 nucleotides of at least one of the nicking agent recognition sequences and each of the two nicking agent recognition sequences being nicked, with (B) a sequence-specific topoisomerase and (C) a second polynucleotide having 5′ hydroxyl on each end; and (II) transforming the mixture into a host organism, thereby cloning the second polynucleotide. Formation or purification of a DNA-protein adduct prior to the addition of the second polynucleotide is not required. Also provided are vector sequences to facilitate performance of the methods and methods for modifying a vector of interest to render it useful in the disclosed methods. 121.-. (canceled)22. A composition comprising(i) a first polynucleotide having a first strand and a second strand, wherein said first polynucleotide is a first vector that comprises (a) a stuffer sequence, (b) a first topoisomerase recognition sequence and a second topoisomerase recognition sequence, (c) a first nicking agent recognition sequence and a second nicking agent recognition sequence, and a (d) selectable marker,(ii) a sequence-specific topoisomerase that reversibly cleaves a single strand of double stranded nucleic acid of the first polynucleotide in the presence of said first topoisomerase recognition sequence or said second topoisomerase recognition sequence, and(iii) a second polynucleotide, whereinthe first topoisomerase recognition sequence is in within fifty nucleotides of the first nicking agent recognition sequence,the first topoisomerase recognition sequence is in said first strand and said first nicking agent recognition sequence directs a nicking agent to ...

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Номер записи: 76

16-05-2013 дата публикации

Device for automatically analyzing nucleic acid

Номер: US20130122576A1

Автор: Oh Won Kwon

Принадлежит: Korea Institute of Machinery and Materials KIMM

An apparatus for automatically analyzing a nucleic acid includes: a sample preprocessing device including a plurality of chambers in which reagents mixed with a sample are accommodated according to sample preprocessing process order for extracting a nucleic acid from the sample; and a nucleic amplifying and detecting device connected with the sample preprocessing device to receive the nucleic acid extracted from the sample.

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Номер записи: 77

16-05-2013 дата публикации

SYNTHETIC CONSTRUCTS FOR POLYNUCLEOTIDE & PROTEIN EXPRESSION

Номер: US20130123346A1

Автор: Bravo Soraya, Cafferata Eduardo, Cruzat Fernando, De Ferrari Giancarlo, Montecino Martin Alejandro, Nunez Hovivera Felipe Javier, Podhajcer Osva Ldo Luis

Принадлежит:

The present invention features, inter alia, nucleic acid constructs that include nucleotide sequences for regulating the expression of a sequence of interest. The sequences in the construct are not naturally associated with one another (i.e., they are heterologous), and they include an enhancer comprising response elements (e.g. Tcf sites) and nucleosome positioning regions. The enhancer can be operably linked to a promoter (e.g., a human REG1A-571 promoter) that drives the expression of a sequence of interest. Also included are vectors comprising these constructs, host cells, kits, pharmaceutical formulations, and methods of treating patients with cancer. 1. A nucleic acid construct comprising an enhancer , wherein the enhancer comprises a first sequence having at least one response element and a second sequence having at least one nucleosome positioning region.2. The construct of claim 1 , wherein the first sequence comprises at least one Tcf site.3. The construct of claim 2 , wherein the first sequence comprises 2-10 Tcf sites.4. The construct of claim 3 , wherein the first sequence comprises four Tcf sites.54. The construct of any of - claims 1 , wherein the first sequence comprises SEQ ID NO:1 or a biologically active fragment or other variant thereof.6. The construct of claim 5 , wherein the biologically active fragment is residues 7-78 of SEQ ID NO:1.7. The construct of claim 5 , wherein the biologically active variant is a sequence that is at least 80% identical to SEQ ID NO:1 or to residues 7-78 of SEQ ID NO:1.87. The construct of any of - claims 2 , wherein the Tcf site is identical to SEQ ID NO:13.98. The construct of any of - claims 1 , wherein the second sequence comprises SEQ ID NO:2 or a biologically active fragment or other variant thereof.10. The construct of claim 9 , wherein the biologically active fragment is residues 7-190 of SEQ ID NO:2.11. The construct of claim 9 , wherein the biologically active variant is a sequence that is at least 80% ...

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Номер записи: 78

23-05-2013 дата публикации

METHOD OF SYNTHESIZING A SUPPRESSOR tRNA, DNA CONSTRUCT AND USE THEREOF FOR PRODUCING A NON-NATURAL AMINO ACID-INCORPORATED PROTEIN

Номер: US20130130313A1

Автор: Hino Nobumasa, KOBAYASHI Takatsugu, Mukai Takahito, Sakamoto Kensaku, Yokoyama Shigeyuki

Принадлежит: RIKEN

There are provided a DNA construct comprising a suppressor tRNA gene of a non-eukaryote containing no internal promoter functioning in a eukaryotic cell, and a eukaryotic or bacteriophage promoter linked at the 5′ end of the tRNA gene, a method for synthesizing a suppressor tRNA by using the DNA construct, and a process for producing protein incorporating a non-natural amino acid by using the same. 1Escherichia coli. A DNA construct comprising a suppressor tRNA gene originating from , wherein said tRNA contains no internal promoter sequence that functions in an eukaryotic cell , and has an eukaryotic promoter linked to the 5′ end of said tRNA gene.2. The DNA construct of claim 1 , wherein said tRNA gene is a suppressor tyrosine tRNA gene.3. The DNA construct of claim 1 , further comprising a transcription terminator sequence linked to the 3′ end of said tRNA gene.4. The DNA construct of claim 1 , wherein said eukaryotic promoter is RNA polymerase II or III.5. The DNA construct of claim 4 , wherein said RNA polymerase II is a promoter of a U1 snRNA gene.6. The DNA construct of claim 4 , wherein said RNA polymerase III is a promoter of a U6 snRNA gene.7. The DNA construct of claim 6 , wherein said promoter of a U6 snRNA gene has the nucleotide sequence set forth in SEQ ID NO:3 or a nucleotide sequence that is at least 30% claim 6 , 50% claim 6 , 70% claim 6 , 90% or 95% identical thereto claim 6 , and induces transcription by RNA polymerase III in a mammalian cell.8. A method of synthesizing a suppressor tRNA comprising causing the DNA construct of to undergo transcription in an eukaryotic cell.9. A recombinant eukaryotic cell that is transformed or transfected by the DNA construct of .10Escherichia coli. A process for producing a non-natural amino acid incorporated-protein comprising: expressing claim 1 , in the presence of the non-natural amino acid claim 1 , in an eukaryotic cell comprising a DNA construct comprising a suppressor tRNA gene originating from claim 1 ...

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Номер записи: 79

23-05-2013 дата публикации

BIOSYNTHESIS OF CAFFEIC ACID AND CAFFEIC ACID DERIVATIVES BY RECOMBINANT MICROORGANISMS

Номер: US20130130340A1

Автор: LIN Yuheng, Yan Yajun

Принадлежит:

Microorganisms are genetically engineered to synthesize caffeic acid from simple carbon sources via a tyrosine intermediate by means of a dual pathway that utilizes both endogenous and engineered enzymatic activities. 1. A genetically engineered cell comprising p-coumarate 3-hydroxylase (C3H) enzyme activity , wherein said cell has been metabolically engineered to express or overexpress a tyrosine ammonia lyase (TAL).2. The genetically engineered cell of wherein the p-coumarate 3-hydroxylase activity is provided by an endogenous 4-hydroxyphenylacetate 3-hydroxylase (4HPA3H) enzyme or enzyme complex.3. The genetically engineered cell of or which has been further metabolically engineered to overexpress a 4HPA3H enzyme or enzyme complex.4. The genetically engineered cell of further comprising a metabolic pathway for the biosynthesis of tyrosine.5. The genetically engineered cell of which has been further metabolically engineered to overproduce tyrosine compared to a wild-type cell.6. The genetically engineered cell of which has been further metabolically engineered to reduce or eliminate feedback inhibition of tyrosine biosynthesis.7. The genetically engineered cell of any of or which has been further metabolically engineered to redirect carbon flow toward tyrosine biosynthesis.8. The genetically engineered cell of which is a bacterial cell.9Escherichia coli. The genetically engineered cell of which is an cell.10. The genetically engineered cell of comprising at least one vector operably encoding the tyrosine ammonia lyase (TAL).11. The genetically engineered cell of comprising at least one vector operably encoding an enzyme having p-coumarate 3-hydroxylase (C3H) activity.12. The genetically engineered cell of wherein a single vector encodes both the tyrosine ammonia lyase (TAL) and the enzyme having p-coumarate 3-hydroxylase (C3H) activity.13. The genetically engineered cell of comprising a first vector encoding the tyrosine ammonia lyase (TAL) and a second vector ...

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Номер записи: 80

23-05-2013 дата публикации

MICROORGANISM VARIANTS HAVING HYDROCARBON PRODUCING ABILITY AND METHOD FOR PRODUCING HYDROCARBON USING THE SAME

Номер: US20130130344A1

Автор: CHOI Yong Jun, LEE Sang Yup

Принадлежит: KOREA ADVANCED INSTITUTE OF SCIENCE AND TECHNOLOGY

The present invention relates to a microorganism variant having the ability to produce hydrocarbons, including alkane, and a method of producing hydrocarbons, including alkane, using the same, and more particularly, to a microorganism variant obtained by introducing a gene encoding an enzyme converting fatty acyl-acp to free fatty acid, a gene encoding an enzyme converting free fatty acid to fatty acyl-CoA, a gene encoding an enzyme converting fatty acyl-CoA to fatty aldehyde and a gene encoding an enzyme converting fatty aldehyde to alkane into a microorganism improved so as to be suitable for the production of hydrocarbons, including alkane, and a method of producing hydrocarbons, including alkane, using the same. The microorganism variant of the present invention has high potential to be used to improve strains by additional metabolic flux engineering, and thus is useful for the industrial production of hydrocarbons, including alkane. 1. A microorganism variant having the ability to produce hydrocarbons selected from the group consisting of alkanes , alkenes , alkynes , and aromatic hydrocarbons , wherein a gene encoding acyl coenzyme A dehydrogenase and a gene encoding a DNA-binding transcriptional dual regulator are deleted or attenuated; and a gene encoding an enzyme converting fatty acyl-acp to free fatty acid , a gene encoding an enzyme converting free fatty acid to fatty acyl-CoA , a gene encoding an enzyme converting fatty acyl-CoA to fatty aldehyde , and a gene encoding an enzyme converting fatty aldehyde to alkane are introduced or amplified.2. The microorganism variant of claim 1 , wherein the native promoter of the gene encoding acyl-CoA synthetase and the attenuator-containing native promoter of the gene encoding the fatty acid outer membrane transporter are further substituted with a strong promoter in the microorganism variant claim 1 , and a gene encoding a short-chain fatty acid outer membrane transporter is further introduced into the ...

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Номер записи: 81

23-05-2013 дата публикации

ENHANCED EXPRESSION AND STABILITY REGIONS

Номер: US20130130372A1

Автор: BABB Robert, Chen Gang, Fandl James P.

Принадлежит: Regeneron Pharmaceuticals, Inc.

Expression-enhancing nucleotide sequences for expression in eukaryotic systems are provided that allow for enhanced and stable expression of recombinant proteins in eukaryotic cells. Enhanced expression and stability regions (EESYRs) are provided for expression of a gene of interest in a eukaryotic cell. Chromosomal loci, sequences, and vectors are provided for enhanced and stable expression of genes in eukaryotic cells. 1. An isolated cell comprising an expression-enhancing sequence selected from the group consisting of SEQ ID NOs:1-4 , a gene of interest (GOI) operably linked to the expression-enhancing sequence , and at least one recombinase recognition site immediately adjacent to the GOI.2. The isolated cell of claim 1 , wherein the GOI encodes a protein selected from an immunoglobulin or an antigen-binding fragment thereof claim 1 , and a receptor or ligand-binding fragment thereof.3. The isolated cell of claim 2 , wherein the immunoglobulin is selected from an antibody light chain or antigen-binding fragment thereof claim 2 , an antibody heavy chain or antigen-binding fragment thereof claim 2 , or an Fc fusion protein.4. The isolated cell of claim 1 , wherein the at least one recombinase recognition site is selected from the group consisting of a LoxP site claim 1 , a Lox511 site claim 1 , a Lox2272 claim 1 , and a Frt site.5. The isolated cell of claim 1 , wherein the GOI is immediately adjacent and 5′ of the recombinase recognition site claim 1 , and further comprising a second recombinase recognition site immediately adjacent and 3′ of the GOI.6. The isolated cell of claim 5 , further comprising a second GOI immediately adjacent and 3′ of the second recombinase recognition site.7. The isolated cell of claim 6 , further comprising a third recombinase recognition site immediately adjacent and 3′ of the second GOI.8. The isolated cell of claim 7 , further comprising at least one marker gene between the second recombinase recognition site and the second GOI.9. ...

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Номер записи: 82

23-05-2013 дата публикации

SURFACE MARKERS AND USES THEREOF FOR RAPID STABLE CELL LINE GENERATION AND GENE AMPLIFICATION

Номер: US20130130385A1

Автор: Tu Hua

Принадлежит:

The present invention provides methods of producing recombinant cells, methods of large scale production of a gene expression product (such as protein), and methods of establishing a stable cell line using the surface markers. Also provided are expression vectors encoding the surface markers and cells comprising the expression vectors. Further provided are gene expression products (such as proteins) and cells obtained using methods described herein, as well as kits useful for carrying out methods described herein. 1. A method of producing a recombinant cell , comprising: exposing a population of host cells comprising: i) a first nucleic acid sequence comprising a gene of interest , and ii) a second nucleic acid sequence comprising a coding sequence for a surface marker to a separation means that recognizes the surface marker , wherein cells recognized by the separation means can be separated from the rest of the cells.2. A method of large scale production of a gene expression product , comprising exposing a population of host cells comprising: i) a first nucleic acid sequence comprising a gene of interest , and ii) a second nucleic acid sequence comprising a coding sequence for a surface marker to a separation means that recognizes the surface marker , wherein cells recognized by the separation means can be separated from the rest of the cells.3. A method of establishing a stable cell line , comprising exposing a population of host cells comprising: i) a first nucleic acid sequence comprising a gene of interest , and ii) a second nucleic acid sequence comprising a coding sequence for a surface marker to a separation means that recognizes the surface marker , wherein cells recognized by the separation means can be separated from the rest of the cells.4. The method of claim 2 , wherein the surface marker comprises a tag sequence and a transmembrane domain.5. The method of claim 2 , wherein the surface marker comprises a tag sequence and a membrane anchoring region.6. ...

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Номер записи: 83

23-05-2013 дата публикации

MODIFIED DENDRITIC CELLS HAVING ENHANCED SURVIVAL AND IMMUNOGENICITY AND RELATED COMPOSITIONS AND METHODS

Номер: US20130130386A1

Автор: Lapteva Natalia, PARK Dongsu, SPENCER David

Принадлежит:

Modified antigen presenting cells provided herein have improved lifespan and immunogenicity compared to unmodified antigen presenting cells, and are useful for immunotherapy. The modified antigen presenting cells express an altered protein kinase, referred to herein as “Akt.” The altered Akt associates with the cell membrane with greater frequency than unaltered Akt, and is referred to herein as “membrane-targeted Akt.” 1. A method for preparing a modified dendritic cell , which comprises:transferring a polynucleotide into a dendritic cell, wherein the polynucleotide encodes a membrane-targeted Akt protein comprising a dual acylation region and a mammalian Akt region, wherein the mammalian Akt region comprises the amino acid sequence of SEQ ID NO: 6, wherein the membrane-targeted Akt protein is expressed in the dendritic cell and the modified dendritic cell survives longer than dendritic cells that do not express the protein.2. A method for preparing a modified dendritic cell , which comprises:transferring a polynucleotide into a dendritic cell, wherein the polynucleotide encodes a membrane-targeted Akt protein comprising a dual acylation region and a mammalian Akt region, wherein the mammalian Akt region consists of the amino acid sequence of SEQ ID NO: 6, wherein the membrane-targeted Akt protein is expressed in the dendritic cell and the modified dendritic cell survives longer than dendritic cells that do not express the protein.3. The method of claim 1 , wherein the acylation region comprises the amino acid sequence of SEQ ID NO: 8.4. The method of claim 1 , wherein the acylation region consists of the amino acid sequence of SEQ ID NO: 8.5. The method of claim 1 , wherein the polynucleotide is from a virus.6. The method of claim 5 , wherein the dendritic cell is contacted with a virus that contains the polynucleotide.7. The method of claim 1 , wherein the polynucleotide comprises a constitutively active promoter operably linked to the polynucleotide that encodes ...

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Номер записи: 84

23-05-2013 дата публикации

METHOD FOR GENERATING INDUCED PLURIPOTENT STEM CELLS FROM KERATINOCYTES DERIVED FROM PLUCKED HAIR FOLLICLES

Номер: US20130130387A1

Автор: Itskovitz-Eldor Joseph, Novak-Petraro Atara, Shtrichman Ronit

Принадлежит: TECHNION RESEARCH & DEVELOPMENT FOUNDATION LIMITED

A method for generating induced pluripotent stem (iPS) cells from isolated hair follicles is disclosed. The method comprises: 1. A method for generating induced pluripotent stem (iPS) cells from isolated hair follicles , the method comprising:a. culturing isolated hair follicle keratinocytes on a layer of feeder cells, wherein said isolated keratinocytes are generated by dissociating cells of the hair follicle, so as to generate colonies of hair follicle keratinocytes;b. detaching said colonies of hair follicle keratinocytes from said feeder cells so as to generate detached keratinocytes;c. infecting said detached keratinocytes with a virus comprising a nucleic acid molecule encoding at least OCT4 and SOX2 and dedifferentiation factors so as to generate infected keratinocytes; andd. culturing said infected keratinocytes on a layer of feeder cells in a culture medium until iPS cells are formed, thereby generating iPS cells.2. The method of claim 1 , wherein said nucleic acid molecule further encodes KLF4 and/or C-MYC.3. The method of claim 1 , wherein said colonies comprise between 20-30 hair follicle keratinocytes.4. The method of claim 1 , wherein said isolated hair follicle keratinocytes are in contact with said virus for less than 2 hours.5. The method of claim 1 , wherein said isolated hair follicle keratinocytes are in contact with said virus for less than one hour.6. The method of claim 1 , wherein said virus is a lentivirus.7. The method of claim 1 , wherein said isolated hair follicle keratinocytes are not passaged for more than 3 passages.8. The method of claim 1 , wherein said isolated hair follicle keratinocytes are passaged for 2-3 passages.9. The method of claim 1 , wherein said dissociating is effected using trypsin.10. The method of claim 1 , wherein said infecting is effected during centrifugation at a centrifugal force of about 200 g to about 1000 g.11. The method of claim 1 , wherein said infecting is effected at a temperature between 25° C.-37° C. ...

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Номер записи: 85

23-05-2013 дата публикации

Methods of Modifying Eurakyotic Cells

Номер: US20130130388A1

Автор: MURPHY Andrew J., YANCOPOULOS George D.

Принадлежит: Regeneron Pharmaceuticals, Inc.

A method for engineering and utilizing large DNA vectors to target, via homologous recombination, and modify, in any desirable fashion, endogenous genes and chromosomal loci in eukaryotic cells. These large DNA targeting vectors for eukaryotic cells, termed LTVECs, are derived from fragments of cloned genomic DNA larger than those typically used by other approaches intended to perform homologous targeting in eukaryotic cells. Also provided is a rapid and convenient method of detecting eukaryotic cells in which the LTVEC has correctly targeted and modified the desired endogenous gene(s) or chromosomal locus (loci) as well as the use of these cells to generate organisms bearing the genetic modification. 1. A method for targeted insertion of non-endogenous DNA at a desired position in a non-human genome of a eukaryotic cell , comprisingselecting a bacterial artificial chromosome (BAC) clone comprising a large genomic DNA fragment comprising a DNA sequence of interest;employing the BAC clone to generate a large targeting vector comprising the large genomic DNA fragment comprising the DNA sequence of interest;employing the large targeting vector to modify a non-human genome at a desired position with the large targeting vector.2. The method of claim 1 , wherein the non-endogenous DNA is homologous to DNA in the non-human genome at the desired position in the non-human genome.3. The method of claim 1 , wherein the non-endogenous DNA is orthologous to DNA in the non-human genome at the desired position in the non-human genome.4. The method of claim 1 , wherein the non-endogenous DNA comprises unrearranged human immunoglobulin sequences and the desired position is a non-human immunoglobulin locus.5. The method of claim 1 , wherein the non-human genome is a rodent genome.6. The method of claim 5 , wherein the rodent genome is a mouse genome.7. The method of claim 1 , wherein the eukaryotic cell is a mammalian cell.8. The method of claim 1 , wherein the mammalian cell is a ...

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Номер записи: 86

23-05-2013 дата публикации

Nonviral Gene Delivery Vector Iopamidol, Protamine, Ethiodized Oil Reagent (Viper)

Номер: US20130131154A1

Автор: Higgins Luke J., Hofmann Lawrence V., Hwang Gloria L.

Принадлежит: THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY

Embodiments are related to nonviral gene delivery vectors using only FDA-approved components: iopamidol, protamine, and ethiodized oil. 1. A nonviral gene delivery vector comprising iopamidol , protamine , and ethiodized oil.2. A drug product comprising a nonviral gene delivery vector consisting of plasmid DNA , iopamidol , protamine , and ethiodized oil.3. The vector of further comprising plasmid DNA.4. The vector of further comprising short interfering RNAs (siRNAs) claim 1 , microRNAs (miRNAs) claim 1 , antisense oligonucleotides (ASO) claim 1 , ribozymes claim 1 , or triplex-forming oligonucleotides.5. The vector of claim 1 , wherein the protamine and DNA form a complex having a protamine to DNA mass ratio of between 1:1 to 1000:1.6. The vector of claim 2 , wherein the protamine and DNA form a complex having a protamine to DNA mass ratio of between 1:1 to 1000:1.7. The vector of claim 1 , wherein the concentration of iopamidol is between 1% and 100%.8. The vector of claim 2 , wherein the concentration of iopamidol is between 1% and 100%.9. The vector of claim 1 , wherein the concentration of ethiodized oil is between 1% and 100%.10. The vector of claim 2 , wherein the concentration of ethiodized oil is between 1% and 100%.11. The vector of claim 2 , wherein the amount of plasmid DNA is between 1 ng and 4000 ng.12. The vector of claim 3 , wherein the amount of plasmid DNA is between 1 ng and 4000 ng.13. The vector of claim 2 , wherein the plasmid DNA comprises a tumor suppressor gene claim 2 , encodes a tumor-specific antigen claim 2 , encodes an enzyme capable of converting a prodrug to a cytotoxic drug claim 2 , comprises a cytotoxic or pro-apoptotic gene claim 2 , encodes a cytokine claim 2 , encodes an anti-angiogenic factor claim 2 , encodes UGT1A1 claim 2 , encodes ornithine transcarbamylase claim 2 , encodes factor VIII claim 2 , encodes factor IX claim 2 , or comprises a combination of the Neurod and Btc genes.14. A process for transfecting mammalian ...

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Номер записи: 87

30-05-2013 дата публикации

METHODS AND COMPOSITIONS FOR MODIFICATION OF THE HPRT LOCUS

Номер: US20130137104A1

Автор: Cost Gregory J., Holmes Michael C., Kasahara Noriyuki, Laganiere Josee, Miller Jeffrey C., Paschon David, Rebar Edward J., Urnov Fyodor, ZHANG LEI

Принадлежит:

Nucleases and methods of using these nucleases for modification of an HPRT locus and for increasing the frequency of gene modification at a targeted locus and clones and for generating animals. 1. A non-naturally occurring fusion protein comprising a zinc finger protein that binds to an endogenous hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene and a cleavage domain , wherein the fusion protein modifies the endogenous HPRT gene.2. The fusion protein of claim 2 , wherein the zinc finger protein comprises 4 claim 2 , 5 or 6 zinc finger domains comprising a recognition helix region claim 2 , wherein the zinc finger proteins comprise the recognition helix regions in the order shown in a single row of Table 1.3. A polynucleotide encoding one or more fusion proteins of .4. An isolated cell comprising one or more fusion proteins according to .5. An isolated cell comprising one or more polynucleotides according to .6. The cell of claim 4 , wherein the cell is selected from the group consisting of a T-cell claim 4 , a B-cell or a stem cell.7. The cell of claim 6 , wherein the stem cell is selected from the group consisting of an embryonic stem cell (ESC) claim 6 , an induced pluripotent stem cell (iPSC) claim 6 , a CD34+ hematopoietic stem cell and a hepatic stem cell.8. The cell of claim 5 , wherein the cell is selected from the group consisting of a T-cell claim 5 , a B-cell or a stem cell.9. The cell of claim 8 , wherein the stem cell is selected from the group consisting of an embryonic stem cell (ESC) claim 8 , an induced pluripotent stem cell (iPSC) claim 8 , a CD34+ hematopoietic stem cell and a hepatic stem cell.10. A kit comprising a fusion protein according to .11. A kit comprising a polynucleotide according to .12. A method of cleaving an endogenous HPRT gene in a cell claim 3 , the method comprising:{'claim-ref': {'@idref': 'CLM-00003', 'claim 3'}, 'introducing, into the cell, one or more polynucleotides according to , under conditions such that the ...

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Номер записи: 88

30-05-2013 дата публикации

GENOME EDITING USING TARGETING ENDONUCLEASES AND SINGLE-STRANDED NUCLEIC ACIDS

Номер: US20130137180A1

Автор: Chen Fuqiang, Davis Gregory D., Pruett-Miller Shondra M.

Принадлежит: SIGMA ALDRICH CO, LLC

The present invention provides methods and kits for editing specific chromosomal sequences in cells. In particular, targeting endonucleases and single-stranded nucleic acids are used to edit the chromosomal sequence. 1. A method for editing at least one endogenous chromosomal sequence in a cell , the method comprising:a) introducing into the cell (i) at least one targeting endonuclease or nucleic acid encoding a targeting endonuclease, the targeting endonuclease being able to introduce a double-stranded break at a targeted cleavage site in the chromosomal sequence, and (ii) at least one single-stranded nucleic acid comprising a first region having substantial sequence identity to the chromosomal sequence on one side of the targeted cleavage site; andb) maintaining the cell under conditions such that the double-stranded break introduced by the targeting endonuclease is repaired by a homology-directed process such that the chromosomal sequence is exchanged with the sequence of the single-stranded nucleic acid, thereby editing the chromosomal sequence.2. The method of claim 1 , wherein the single-stranded nucleic acid comprises a second region having substantial sequence identity to the chromosomal sequence on the other side of the targeted cleavage site.3. The method of claim 2 , wherein the single-stranded nucleic acid further comprises a change of at least one nucleotide relative to the chromosomal sequence.4. The method of claim 2 , wherein the single-stranded nucleic acid further comprises an exogenous sequence that is flanked by the first region and the second region.5. The method of claim 1 , wherein the single-stranded nucleic acid comprises a second region having substantial sequence identity to a distal chromosomal sequence that is located upstream or downstream of the targeted cleavage site.6. The method of claim 5 , wherein the single-stranded nucleic acid further comprises an exogenous sequence that is flanked by the first region and the second region.7. ...

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Номер записи: 89

30-05-2013 дата публикации

Sequence tag directed subassembly of short sequencing reads into long sequencing reads

Номер: US20130137588A1

Автор: Emily Turner, Jay Shendure, Joseph Hiatt, Rupali Patwardhan

Принадлежит: UNIVERSITY OF WASHINGTON

The invention provides compositions and methods for preparing DNA sequencing libraries. In particular, the method relates to preparing DNA sequencing libraries from kilobase scale nucleic acids. The invention also provides methods for assembling short read sequencing data into longer contiguous sequences. The method is useful for various applications in genomics, including genome assembly, full length cDNA sequencing, metagenomics, and the analysis of repetitive sequences of assembled genomes.

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Номер записи: 90

30-05-2013 дата публикации

Non-Emulsion Methods and Masked Biomolecules

Номер: US20130137606A1

Автор: Gordon Steven, Stetson Daniel

Принадлежит:

The present invention relates to methods and devices for amplifying nucleic acid, and, in particular, amplifying so as to generate products on a surface without the use of emulsions. In a preferred embodiment, a plurality of groups of amplified product are generated on the surface, each group positioned in different (typically predetermined) locations on said surface so as to create an array. 1. A method for making an array , comprising: i) a first element comprising a surface;', 'ii) a second element comprising bottom and top surfaces, said second element further comprising a plurality of channels extending from said bottom to said top surfaces;', 'iii) a third element comprising a surface, and', 'iv) a solution comprising biomolecules, said biomolecules further comprising anchoring molecules attached thereto;, 'a) providingb) contacting the surface of said first element with said solution under conditions such that at least a portion of said biomolecules attach to at least a portion of said surface via said anchoring molecule so as to create a modified surface comprising attached biomolecules; andc) positioning said second element between the surfaces of said first and third elements such that the bottom surface of said second element is on top of said modified surface, so as to create first regions defined by sealed channels, said first regions comprising unmasked attached biomolecules, and second regions comprising masked attached biomolecules, thereby making an array.2. The method of claim 1 , whereby said positioning of step c) removes at least a portion of said solution from said surface of said first element.3. The method of claim 1 , wherein said contacting of step b) results in the entire surface being contacted with said solution.4. The method of claim 1 , wherein said channels of said second element are evenly spaced.5. The method of claim 1 , wherein said attached biomolecules after step b) are attached over substantially the entire surface at a ...

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Номер записи: 91

13-06-2013 дата публикации

G-PROTEIN-CONJUGATED RECEPTOR HAVING ALTERED LIGAND AFFINITY, AND USE THEREOF

Номер: US20130149715A1

Автор: Muramatsu Ikunobu

Принадлежит:

A modified G-protein-coupled receptor (GPCR), having modified ligand affinity is provided by binding a G-protein-coupled receptor to a polypeptide consisting of an amino acid sequence of SEQ ID NO: 1. Furthermore, agonists for or antagonists against the modified GPCR are screened using a transformant in which the modified GPCR has been expressed. This makes it possible to provide a technique for analyzing the function of many putative GPCRs whose entities have not been clarified. 1. A protein complex of a GPCR and a polypeptide ,the polypeptide being:(A) a polypeptide consisting of an amino-acid sequence of SEQ ID NO: 1;(B) a polypeptide (i) consisting of an amino-acid sequence of SEQ ID NO: 1 with a deletion, insertion, substitution, or addition of one or several amino acids, and (ii) having activity to modify ligand affinity of a GPCR with which the polypeptide has formed a complex;(C) a polypeptide encoded by a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2;(D) a polypeptide (i) encoded by a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2 with a deletion, insertion, substitution, or addition of one or several nucleotides, and (ii) having activity to modify ligand affinity of a GPCR with which the polypeptide has formed a complex;(E) a polypeptide (i) encoded by a polynucleotide capable of hybridizing under stringent conditions with a polynucleotide consisting of a sequence complementary to a nucleotide sequence of SEQ ID NO: 2 and (ii) having activity to modify ligand affinity of a GPCR with which the polypeptide has formed a complex; or(F) a polypeptide (i) coded for by a polynucleotide having a sequence identity of 70% or higher with a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2 and (ii) having activity to modify ligand affinity of a GPCR with which the polypeptide has formed a complex.2. The protein complex as set forth in claim 1 , wherein the GPCR is an adrenergic receptor claim 1 , a dopamine ...

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Номер записи: 92

13-06-2013 дата публикации

METHODS FOR MAKING AND USING MOLECULAR SWITCHES INVOLVING CIRCULAR PERMUTATION

Номер: US20130149785A1

Автор: Guntas Gurkan, Ostermeier Marc Alan

Принадлежит: THE JOHNS HOPKINS UNIVERSITY

The invention provides molecular switches which couple external signals to functionality, and combinatorial methods of making and using the same involving circular permutation of nucleic acid and amino acid sequences. The switches according to the invention can be used, for example, to regulate gene transcription, target drug delivery to specific cells, transport drugs intracellularly, control drug release, provide conditionally active proteins, perform metabolic engineering, and modulate cell signaling pathways. Libraries comprising the switches, expression vectors and host cells for expressing the switches are also provided. 1. A method for assembling a fusion molecule , comprising:generating a circular permutation of an insertion sequence; andinserting the insertion sequence into an acceptor sequence.215-. (canceled)16. A method for modulating a cellular activity , comprising:{'claim-ref': {'@idref': 'CLM-00001', 'claim 1'}, 'providing a fusion molecule generated according to the method of to a cell, wherein a change in state of at least the insertion sequence or the acceptor sequence modulates a cellular activity, and wherein the change in state which modulates the cellular activity is coupled to a change in state of the respective other portion of the fusion molecule; and'}changing the state of the respective other portion of the fusion molecule, thereby modulating the cellular activity.17. A method for delivering a bio-effective molecule to a cell , comprising:{'claim-ref': {'@idref': 'CLM-00001', 'claim 1'}, 'providing a fusion molecule associated with a bio-effective molecule generated according to the method of to the cell, the fusion molecule comprising an insertion sequence and an acceptor sequence, wherein either the insertion sequence or the acceptor sequence binds to a cellular marker of a pathological condition and wherein upon binding to the marker, the fusion molecule dissociates from the bio-effective molecule, thereby delivering the molecule to ...

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Номер записи: 93

20-06-2013 дата публикации

METHOD FOR PREPARING HUMAN NEOPLASTICALLY TRANSFORMED CELLS

Номер: US20130157304A1

Автор: Hamada Katsutomo

Принадлежит: TRDIGM & CO., LTD.

A method for preparing neoplastically transformed cells from human-derived cells, including the step of introducing human telomerase catalytic subunit (hTERT) gene, SV40 small T antigen (SV40ST) gene, and an antisense oligonucleotide derived from human 28S rRNA into the human-derived cells. The method for preparing neoplastically transformed cells from human-derived cells can be utilized when a variety of human normal cells are induced to be neoplastically transformed in order to elucidate cancer onset mechanisms, so that the method can be effectively utilized in search of target molecules for a new medicament. 1. A method for preparing a neoplastically transformed cell from a human-derived cell , the method comprising introducing a human telomerase catalytic subunit (hTERT) gene , an SV40 small T antigen (SV40ST) gene , and an antisense oligonucleotide derived from a human 28S rRNA into the human-derived cell.2. The method according to claim 1 , wherein the antisense oligonucleotide derived from a human 28S rRNA isa first oligonucleotide comprising a nucleotide sequence of SEQ ID NO: 1, ora second oligonucleotide comprising a sequence having 70% or more homology to the nucleotide sequence of SEQ ID NO: 1,wherein a neoplastic transformation efficiency of the second oligonucleotide is equivalent to a neoplastic transformation efficiency of the first oligonucleotide.3. The method according to claim 1 , wherein the antisense oligonucleotide derived from a human 28S rRNA isa first oligonucleotide comprising a nucleotide sequence of SEQ ID NO: 2, ora second oligonucleotide comprising a sequence having 70% or more homology to the nucleotide sequence of SEQ ID NO: 2,wherein a neoplastic transformation efficiency of the second oligonucleotide is equivalent to a neoplastic transformation efficiency of the first oligonucleotide.4. The method according to claim 1 , wherein the human-derived cell is a human fibroblast cell.5. A method for introducing a gene that neoplastically ...

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Номер записи: 94

20-06-2013 дата публикации

BIDIRECTIONAL PROMOTER REPORTER VECTOR FOR THE ANALYSIS OF DUAL REGULATORY ELEMENTS

Номер: US20130157308A1

Автор: Polson Amanda, Reisman David

Принадлежит: UNIVERSITY OF SOUTH CAROLINA

A bidirectional expression vector is described that can be utilized to determine the existence and characteristics of bidirectional promoters. The bidirectional expression vector includes two different reporter genes in a head to head (5′ to 5′) arrangement. In addition, the bidirectional expression vector can include a polylinker region located between the heads of the two reporter genes that provides multiple cloning sites for nonexclusive examination of polynucleotide sequences. The vector can also include a splicing site and drug resistance. The bidirectional expression vector can be used to examine a polynucleotide sequence for the presence of divergent regulator regions and, following determination of a bidirectional promoter, can be utilized to further elucidate characteristics of the bidirectional promoter. 1. A bidirectional expression vector comprising a first reporter gene and a second reporter gene , the first and second reporter genes being arranged in a head to head (5′ to 5′) arrangement , the bidirectional expression vector including a polylinker region between the heads of the first and second reporter genes , the bidirectional expression vector including multiple cloning sites within the polylinker region.2renilla. The bidirectional expression vector of claim 1 , wherein the first reporter gene is a gene.3. The bidirectional expression vector of claim 1 , wherein the second reporter gene is a luciferase gene.4. The bidirectional expression vector of claim 1 , the polylinker region including more than one copy of at least one of the restriction sites.6. The bidirectional expression vector of claim 1 , the polylinker region including the following restriction sites: SmaI claim 1 , XmaI claim 1 , KpnI claim 1 , SmaI claim 1 , XmaI claim 1 , and PstI claim 1 , the restriction sites being inserted in the order as provided.7. The bidirectional expression vector of claim 1 , the polylinker region including at least one of each of SmaI claim 1 , KpnI claim ...

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Номер записи: 95

20-06-2013 дата публикации

NOVEL INTERGENIC ELEMENTS FOR ENHANCING GENE EXPRESSION

Номер: US20130157312A1

Автор: Hoeksema Femke, Otte Arie Pieter, Siep Michel, Verhess John Antonius

Принадлежит:

The present invention relates to nucleic acid fragments and constructs comprising genomic nucleotide sequences, which are present upstream of Rb1 and p15C that are associated with intergenic transcription, for the production of a gene product of interest in a eukaryotic, preferably mammalian, host cell in the presence of a stringent selectable marker. The invention further relates to host cells comprising the nucleic acid constructs, to methods for generating the host cells and to methods for producing a gene product of interest using the host cells. 1. A nucleic acid fragment comprising:(a) between 1,000 and 15,000 consecutive nucleotides of a genomic region that is present upstream of the translation initiation site of a vertebrate Rb1 gene; or,(b) at least 1500 consecutive nucleotides from a genomic region that is present from 10.5 to 7 kilobases upstream of the translation initiation site of a vertebrate p15 gene;wherein the fragment, when directly flanking an expression cassette having the nucleotide sequence of SEQ ID NO: 9, both up- and downstream of the expression cassette, produces at least 50% of number of colonies obtained with the same expression cassette when flanked with STARs 7 and 67 upstream of the expression cassette and STAR 7 downstream of the expression cassette SEQ ID NO: 10, when tested under the conditions of Example 1.2. A nucleic acid fragment according to claim 1 , wherein the fragment has at least 80% nucleotide sequence identity over its entire length with at least 1000 consecutive nucleotides of at least one of SEQ ID NO's: 1-4 or 8.3. A nucleic acid fragment according to claim 1 , wherein the fragment is from a human claim 1 , mouse claim 1 , rat claim 1 , hamster claim 1 , bovine claim 1 , chicken claim 1 , dog claim 1 , cavia claim 1 , pig or rabbit genome.4. A nucleic acid fragment according to claim 1 , wherein the nucleic acid fragment is a fragment having at least 80% nucleotide sequence identity over its entire length with a ...

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Номер записи: 96

27-06-2013 дата публикации

Mammalian Cell Lines for Increasing Longevity and Protein Yield from a Cell Culture

Номер: US20130164783A1

Автор: Chang Chien-Hsing, GOLDENBERG David M., Nordstrom Diane, Qu Zhengxing, Rossi Edmund A., Yang Jeng-Dar

Принадлежит: IMMUNOMEDICS, INC.

Disclosed are compositions and methods for increasing the longevity of a cell culture and permitting the increased production of proteins, preferably recombinant proteins, such as antibodies, peptides, enzymes, growth factors, interleukins, interferons, hormones, and vaccines. Cells transfected with an apoptosis-inhibiting gene or vector, such as a triple mutant Bcl-2 gene, can survive longer in culture, resulting in extension of the state and yield of protein biosynthesis. Such transfected cells exhibit maximal cell densities that equal or exceed the maximal density achieved by the parent cell lines. Transfected cells can also be pre-adapted for growth in serum-free medium, greatly decreasing the time required to obtain protein production in serum-free medium. In certain methods, the pre-adapted cells can be used for protein production following transformation under serum-free conditions. The method preferably involves eukaryotic cells, more preferably mammalian cells. 1. A method of producing a mammalian cell line that is pre-adapted for growth and transfection in serum-free medium comprising:a) transfecting a mammalian cell line with a first nucleic acid encoding a mutant Bcl-2 protein comprising T69E, S70E and S87E amino acid substitutions, to produce a Bcl-EEE host cell;b) culturing the Sp-EEE host cell in the presence of methotrexate to amplify the first nucleic acid; andc) adapting the Bcl-EEE host cell to grow in serum-free medium to produce a pre-adapted host cell, wherein the pre-adapted Sp-EEE host cell can be transfected in serum-free medium with a second nucleic acid encoding a recombinant protein.2. The method of claim 1 , further comprising transfecting the pre-adapted host cell in serum-free medium with a second nucleic acid encoding a recombinant protein to produce a recombinant host cell.3. The method of claim 2 , further comprising culturing the recombinant host cell in culture medium to produce the recombinant protein.4. The method of claim 3 , ...

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Номер записи: 97

27-06-2013 дата публикации

Methods and compositions for inactivating glutamine synthetase gene expression

Номер: US20130164785A1

Автор: Jeffrey C. Miller, Pei-Qi Liu

Принадлежит: Sangamo Biosciences Inc

Disclosed herein are methods and compositions for inactivating a glutamine synthetase (GS) gene, using fusion proteins comprising a zinc finger protein and a cleavage domain or cleavage half-domain. Polynucleotides encoding said fusion proteins are also provided, as are cells comprising said polynucleotides and fusion proteins.

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Номер записи: 98

27-06-2013 дата публикации

SELF-DELETING PLASMID

Номер: US20130164790A1

Автор: Cranenburgh Rocky Marc, Leckenby Matthew William

Принадлежит: COBRA BIOLOGICS LTD.

A method of producing a selectable marker gene-free plasmid by culturing a plasmid containing a selectable marker gene flanked by site specific recombinase target sites in a host cell environment incapable of effecting recombination between the site specific recombinase target sites and subsequently culturing the plasmid in another host cell environment which is capable of effecting recombination between the site specific recombinase target sites, so that the selectable marker gene is excised. Uses of plasmids produced by the method for the production of recombinant protein for therapeutic and vaccine purposes, production of therapeutic DNA and DNA vaccines and delivery of recombinant protein and DNA to a patient using live bacterial vectors. 139-. (canceled)40. A method of producing a selectable marker gene-free plasmid comprising the steps of:a) culturing a plasmid containing a selectable marker gene flanked by site specific recombinase target sites selected from Ecdif, cer, psi, pif and mwr in a first host cell environment which is incapable of effecting recombination between the site specific recombinase target sites, wherein the first host cell environment comprises an inactivating mutation in one or more of the genes encoding PepA, ArgR and ArcA; andb) subsequently culturing the plasmid in a second host cell environment which is capable of effecting recombination between the site specific recombinase target sites, such that the selectable marker gene is excised, wherein the second host cell environment contains active versions of PepA and ArgR or ArcA, and comprises a site specific recombinase selected from XerC and XerD.41. The method of further comprising the step of:c) maintaining the selectable marker gene-free plasmid in cell culture.42. The method of further comprising the step of:d) isolating the selectable marker gene-free plasmid from the second host cell environment.43. The method of wherein the first host cell environment and the second host cell ...

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Номер записи: 99

27-06-2013 дата публикации

Expression System

Номер: US20130164823A1

Автор: Baillie Leslie William James, Bullifent Helen Lisa, Flick-Smith Helen Claire, Holden Paula Thomson, MILLER Julie, Titball Richard William, Topping Andrew William, Walker Nicola Jane, WILLIAMSON ETHEL DIANE

Принадлежит:

An immunogenic reagent which produces an immune response which is protective against said reagent comprising one or more polypeptides which together represent up to three domains of the full length Protective Antigen (PA) of or variants of these, and at least one of said domains comprises domain 1 or domain 4 of PA ro a variant thereof. The polypeptides of the immunogenic reagent as well as full length PA are produced by expression from High yields of polypeptide are obtained using this method. Cells, vectors and nucleic acids used in the method are also described and claimed. 1Bacillus anthracis. A recombinant Escherichia coli cell which has been transformed with a nucleic acid which encodes a protective antigen (PA) of or a variant thereof which can produce a protective immune response , wherein the percentage of guanine and cytosine residues within the nucleic acid is in excess of 35%.2Escherichia coli. The recombinant cell of claim 1 , wherein the percentage of guanine and cytosine residues within the nucleic acid is in excess of 45%.3Escherichia coli. The recombinant cell of claim 2 , wherein the percentage of guanine and cytosine residues within the nucleic acid is from 50%-52%.4E. coli. The recombinant cell of claim 1 , wherein the nucleic acid comprises SEQ ID NO:1.5E. coli. The recombinant cell of claim 4 , wherein the nucleic acid is SEQ ID NO:1.6Escherichia coliBacillus anthracis. An transformation vector comprising a nucleic acid which encodes the protective antigen (PA) of or a variant thereof which can produce a protective immune response claim 4 , wherein the percentage of guanine and cytosine residues within the nucleic acid is in excess of 35%.7Bacillus anthracis. A nucleic acid comprising SEQ ID NO:1 or a modified form thereof which encodes the protective antigen (PA) of or a variant thereof Which produces a protective immune response and which has at least 35% GC content.8. The nucleic acid of which is at least 90% identical to SEQ ID NO:1.9. The ...

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Номер записи: 100