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    GENE AMPLIFICATION AND TRANSFECTION METHODS AND REAGENTS RELATED THERETO
    1.
    发明申请
    GENE AMPLIFICATION AND TRANSFECTION METHODS AND REAGENTS RELATED THERETO 审中-公开
    基因扩增和转运方法和相关试剂

    公开(公告)号：US20130164851A1

    公开(公告)日：2013-06-27

    申请号：US13636379

    申请日：2011-03-25

    申请人： Anthony Rossomando ,  Gregory P. Thill ,  Stuart Pollard

    发明人： Anthony Rossomando ,  Gregory P. Thill ,  Stuart Pollard

    IPC分类号： C12N15/85

    CPC分类号： C12N15/1137 ,  C12N15/1138 ,  C12N2310/14

    摘要： Provided herein are methods and compositions for generating a cell line capable of producing a biological product, using a gene amplification based system. Methods and compositions are provided to inhibit endogenous selectable amplifiable marker genes using RNA interference and prevent the selection of false positives during generation of a custom cell line. Such methods improve efficiency of cell line development and do not require the use of specialized substrates or cells lacking the endogenous selectable amplifiable marker gene to negate the effect of endogenously expressed levels of the selectable amplifiable marker gene in cells.

    摘要翻译： 本文提供了使用基于基因扩增的系统产生能够产生生物产物的细胞系的方法和组合物。 提供方法和组合物以使用RNA干扰来抑制内源性选择性可扩增标记基因，并防止在产生定制细胞系期间选择假阳性。 这样的方法提高了细胞系发育的效率，并且不需要使用缺乏内源可选择可扩增标记基因的特异性底物或细胞来消除细胞内可选择可扩增标记基因的内源表达水平的作用。

    Expression of human serum albumin in Pichia pastoris
    3.
    发明授权
    Expression of human serum albumin in Pichia pastoris 失效
    人血清白蛋白在毕赤酵母中的表达

    公开(公告)号：US5707828A

    公开(公告)日：1998-01-13

    申请号：US433037

    申请日：1995-05-03

    申请人： Kotikanyadan Sreekrishna ,  Juerg F. Tschopp ,  Gregory P. Thill ,  Russell A. Brierley ,  Kathryn A. Barr

    发明人： Kotikanyadan Sreekrishna ,  Juerg F. Tschopp ,  Gregory P. Thill ,  Russell A. Brierley ,  Kathryn A. Barr

    IPC分类号： C12N15/09 ,  C07K14/765 ,  C12N1/15 ,  C12N1/19 ,  C12N15/14 ,  C12N15/81 ,  C12P21/02 ,  C12R1/84 ,  C12P21/06 ,  C12N15/00 ,  C12N15/63

    CPC分类号： C12N15/815 ,  C07K14/765

    摘要： The present invention is directed to expression cassettes comprising a 5' regulatory region from at least one of the Pichia pastoris AOXI gene, p40 gene, DASI gene or HIS4 gene, operably linked to an HSA structural gene including the HSA signal sequence. The HSA structural gene has a translational start codon within 0 to 11 deoxyribonucleotides from the 5' end of the HSA structural gene and is operably linked to a 3' termination sequence. Further, the adenine and thymine content of the intervening deoxyribonucleotides is in the range from about 55 to about 64%. The expression cassette may be an autonomously replicating vector or an integrative vector. Other embodiments of the present invention include Pichia pastoris strains transformed with the HSA expression cassettes and processes for secretion of HSA using the expression cassettes.

    摘要翻译： 本发明涉及包含与包含HSA信号序列的HSA结构基因可操作地连接的至少一种巴斯德毕赤酵母AOXI基因，p40基因，DASI基因或HIS4基因的5'调节区的表达盒。 HSA结构基因具有来自HSA结构基因5'末端的0至11个脱氧核糖核苷酸之间的翻译起始密码子，并且可操作地连接到3'终止序列。 此外，介入的脱氧核糖核苷酸的腺嘌呤和胸腺嘧啶含量在约55至约64％的范围内。 表达盒可以是自主复制载体或整合载体。 本发明的其他实施方案包括用HSA表达盒转化的巴斯德毕赤酵母菌株和使用表达盒分泌HSA的方法。

    Production of Bacillus entomotoxins in methylotrophic yeast
    5.
    发明授权
    Production of Bacillus entomotoxins in methylotrophic yeast 失效
    甲基营养酵母中芽孢杆菌毒素的生产

    公开(公告)号：US5827684A

    公开(公告)日：1998-10-27

    申请号：US231342

    申请日：1994-04-19

    申请人： Kotikanyadanam Sreekrishna ,  William D. Prevatt ,  Gregory P. Thill ,  Geneva R. Davis ,  Patricia Koutz ,  Kathryn A. Barr ,  Sharon A. Hopkins

    发明人： Kotikanyadanam Sreekrishna ,  William D. Prevatt ,  Gregory P. Thill ,  Geneva R. Davis ,  Patricia Koutz ,  Kathryn A. Barr ,  Sharon A. Hopkins

    IPC分类号： A01N63/00 ,  A01N63/02 ,  A01N63/04 ,  C07K14/32 ,  C12N1/19 ,  C12N15/09 ,  C12N15/81 ,  C12P21/02 ,  C12R1/07 ,  C12R1/84 ,  C12N15/32 ,  C12N15/63 ,  C12N21/02

    CPC分类号： C12N15/815 ,  A01N63/04 ,  C07K14/32

    摘要： A method for producing one or more Bacillus toxin polypeptides by culturing methylotrophic yeast cells which have a gene(s) capable of expressing the Bacillus toxin polypeptide(s) in such cells under conditions that the gene(s) is/are transcribed is provided. The toxin polypeptide encoding segment of the gene(s) has a G+C content of about 40%-55%, and preferably comprises methylotrophic yeast codons. The preferred species of yeast for expressing such synthetic Bacillus toxin gene(s) is Pichia pastoris. Bacillus toxin polypeptides encoded by synthetic genes are expressed at high levels in transformed methylotrophic yeast cells. The toxin expressing cells may be administered as live cells or heat-killed whole cells to provide an insecticidal composition for killing susceptible insect larvae. Also provided by the present invention are DNAs capable of transforming methylotrophic yeast to express one or more Bacillus toxin polypeptides, cultures of such yeast cells transformed with such DNAs and novel Bacillus toxin polypeptides made by the method of the invention. The transformed yeast cells of the present invention are readily ingested as food by insect larvae which are susceptible to the toxin polypeptides.

    摘要翻译： 提供了一种通过培养具有能够在基因被转录的条件下能够在这些细胞中表达芽孢杆菌毒素多肽的基因的甲基营养酵母细胞产生一种或多种芽孢杆菌毒素多肽的方法。 编码基因片段的毒素多肽的G + C含量为约40％-55％，优选包含甲基营养酵母密码子。 用于表达这种合成芽孢杆菌毒素基因的酵母的优选种类是巴斯德毕赤酵母。 由合成基因编码的芽孢杆菌毒素多肽在转化的甲基营养酵母细胞中以高水平表达。 表达毒素的细胞可以作为活细胞或热灭活的全细胞施用，以提供用于杀灭易感昆虫幼虫的杀虫组合物。 本发明还提供能够转化甲基营养酵母以表达一种或多种芽孢杆菌毒素多肽的DNA，用这种DNA转化的这种酵母细胞的培养物和通过本发明的方法制备的新的芽孢杆菌毒素多肽。 本发明的转化酵母细胞易于被毒素多肽易感的昆虫幼虫摄取为食物。