公开(公告)号：US09309519B2

公开(公告)日：2016-04-12

申请号：US13255004

申请日：2010-03-30

申请人： MyungSam Cho ,  Min Seok Chang ,  Jong-Mook Kim ,  HyunJoo Lee ,  Yoo Cheol Song ,  ManSu Kim

发明人： MyungSam Cho ,  Min Seok Chang ,  Jong-Mook Kim ,  HyunJoo Lee ,  Yoo Cheol Song ,  ManSu Kim

IPC分类号： C12N15/65 ,  C12N15/67 ,  C12N15/10 ,  C12N15/85 ,  C12P21/02 ,  C12N15/69

CPC分类号： C12N15/65 ,  C12N15/1086 ,  C12N15/69 ,  C12N15/85 ,  C12N2800/24 ,  C12N2830/46 ,  C12N2830/50 ,  C12P21/02

摘要： The present invention relates to a method of selecting high producer clones by using an expression vector, the expression vector comprising: (i) a gene expression cassette comprising a selectable marker gene to which polyA has been inoperably linked; and (ii) a gene expression cassette which encodes a recombinant protein of interest and to which polyA has been operably linked. According to the invention, high producer clones can be selected from cell populations at least 10 times fewer than in the existing methods of selecting cell lines. Particularly, high producer clones can be selected using a low concentration of MTX compared to a conventional stepwise gene amplification strategy which comprises carrying out multiple amplification steps while increasing the concentration of MTX. Accordingly, the development period of cell lines can be shortened and the labor and cost required for selection of high-productivity cell clones can be reduced, whereby more efficient production of proteins is possible even when general selectable marker genes other than MTX are used.

摘要翻译： 本发明涉及通过使用表达载体选择高生产者克隆的方法，所述表达载体包含：（i）包含聚A不可操作连接的选择标记基因的基因表达盒; 和（ii）编码目的重组蛋白质并且polyA已被可操作地连接的基因表达盒。 根据本发明，可以从细胞群体中选择高生产者克隆比选择细胞系的现有方法的至少10倍。 特别地，与传统的逐步基因扩增策略相比，可以使用低浓度的MTX来选择高生产者克隆，其包括在增加MTX浓度的同时进行多个扩增步骤。 因此，可以缩短细胞系的发育期，并且可以降低选择高生产率细胞克隆所需的劳动力和成本，从而即使使用MTX以外的一般可选择标记基因，蛋白质的更高效生产也是可能的。

公开(公告)号：US20120009682A1

公开(公告)日：2012-01-12

申请号：US13255004

申请日：2010-03-30

申请人： MyungSam Cho ,  Min Seok Chang ,  Jong-Mook Kim ,  HyunJoo Lee ,  Yoo Cheol Song ,  ManSu Kim

发明人： MyungSam Cho ,  Min Seok Chang ,  Jong-Mook Kim ,  HyunJoo Lee ,  Yoo Cheol Song ,  ManSu Kim

IPC分类号： C12N15/64 ,  C12N5/10 ,  C12N5/16 ,  C12N15/63

CPC分类号： C12N15/65 ,  C12N15/1086 ,  C12N15/69 ,  C12N15/85 ,  C12N2800/24 ,  C12N2830/46 ,  C12N2830/50 ,  C12P21/02

摘要： The present invention relates to a method of selecting high producer clones by using an expression vector, the expression vector comprising: (i) a gene expression cassette comprising a selectable marker gene to which polyA has been inoperably linked; and (ii) a gene expression cassette which encodes a recombinant protein of interest and to which polyA has been operably linked. According to the invention, high producer clones can be selected from cell populations at least 10 times fewer than in the existing methods of selecting cell lines. Particularly, high producer clones can be selected using a low concentration of MTX compared to a conventional stepwise gene amplification strategy which comprises carrying out multiple amplification steps while increasing the concentration of MTX. Accordingly, the development period of cell lines can be shortened and the labor and cost required for selection of high-productivity cell clones can be reduced, whereby more efficient production of proteins is possible even when general selectable marker genes other than MTX are used.

摘要翻译： 本发明涉及通过使用表达载体选择高生产者克隆的方法，所述表达载体包含：（i）包含聚A不可操作连接的选择标记基因的基因表达盒; 和（ii）编码目的重组蛋白质并且polyA已被可操作地连接的基因表达盒。 根据本发明，可以从细胞群体中选择高生产者克隆比选择细胞系的现有方法的至少10倍。 特别地，与传统的逐步基因扩增策略相比，可以使用低浓度的MTX来选择高生产者克隆，其包括在增加MTX浓度的同时进行多个扩增步骤。 因此，可以缩短细胞系的发育期，并且可以降低选择高生产率细胞克隆所需的劳动力和成本，从而即使使用MTX以外的一般可选择标记基因，蛋白质的更高效生产也是可能的。