基本信息:
- 专利标题： Regulated vectors for controlling DNA hypermutability in eukaryotic cells
- 专利标题（中）：用于控制真核细胞中DNA超可变性的调控载体
- 申请号：EP09155285.1 申请日：2003-02-21
- 公开(公告)号：EP2110440A2 公开(公告)日：2009-10-21
- 发明人： Grasso, Luigi , Nicholaides, Nicholas C , Sass, Philip M
- 申请人： Morphotek, Inc.
- 申请人地址： 210 Welsh Pool Road Exton, PA 19341 US
- 专利权人： Morphotek, Inc.
- 当前专利权人： Morphotek, Inc.
- 当前专利权人地址： 210 Welsh Pool Road Exton, PA 19341 US
- 代理机构： Eddowes, Simon
- 优先权： US358602P 20020221
- 主分类号： C12Q1/68
- IPC分类号： C12Q1/68 ; C12N15/85
摘要：
1. The present invention provides a method for producing an isolated, genetically stable somatic cell line with a new phenotype, the method comprising the steps of:
a) culturing a recombinant host somatic cell comprising a vector, said vector comprising a constitutively active promoter operatively linked to a sequence encoding a dominant negative allele of a mismatch repair gene, an internal ribosome entry site, and a negative selection marker, under conditions for the expression of the dominant negative allele, rendering the recombinant host somatic cell hypermutable and thereby producing a library of cells;
b) selecting for clones from the cell library exhibiting new phenotypes;
c) from the clones selected in step (b), negatively selecting for clones no longer expressing the dominant negative allele of a mismatch repair gene, rendering the resulting subclones genetically stable; and
d) expanding a genetically stable subclone of step (c) to generate said genetically stable cell line.
摘要（中）：
a) culturing a recombinant host somatic cell comprising a vector, said vector comprising a constitutively active promoter operatively linked to a sequence encoding a dominant negative allele of a mismatch repair gene, an internal ribosome entry site, and a negative selection marker, under conditions for the expression of the dominant negative allele, rendering the recombinant host somatic cell hypermutable and thereby producing a library of cells;
b) selecting for clones from the cell library exhibiting new phenotypes;
c) from the clones selected in step (b), negatively selecting for clones no longer expressing the dominant negative allele of a mismatch repair gene, rendering the resulting subclones genetically stable; and
d) expanding a genetically stable subclone of step (c) to generate said genetically stable cell line.
1.本发明提供了产生具有新表型的分离的，遗传稳定的体细胞系的方法，所述方法包括以下步骤：a）培养包含载体的重组宿主体细胞，所述载体在操作上包含组成型活性启动子 在显性失活等位基因表达的条件下，与编码错配修复基因的显性失活等位基因，内部核糖体进入位点和阴性选择标记的序列相连，使重组宿主体细胞超可变，从而产生文库 细胞; b）从展示新表型的细胞文库中选择克隆; c）从步骤（b）中选择的克隆中，阴性选择不再表达错配修复基因的显性失活等位基因的克隆，使得到的亚克隆遗传稳定; 和d）扩增步骤（c）的遗传稳定的亚克隆以产生所述遗传稳定的细胞系。