PRODUCTION OF HOMOZYGOUS MUTANT ES CELLS WITH A SINGLE TARGETING CONSTRUCT
PRODUCTION OF HOMOZYGOUS MUTANT ES CELLS WITH A SINGLE TARGETING CONSTRUCT
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DOI：
10.1128/mcb.12.5.2391
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发表时间：
1992-05-01
影响因子：
5.3
通讯作者：
SEIDMAN, JG
中科院分区：
文献类型：
--
作者：
MORTENSEN, RM;CONNER, DA;SEIDMAN, JG
我们已经开发了一种简单的方法，用于生产胚胎干（ES）细胞系，其中两个等位基因已被灭活的同源重组，这需要一个单一的靶向构建体。创建了四种不同的ES细胞系，其对于编码两种鸟嘌呤核苷酸结合蛋白亚基α（i2）和α（i3）、T细胞受体α和β-心肌肌球蛋白重链的基因是杂合的。当这些杂合细胞在高浓度或G418中生长时，许多存活的细胞对于靶向等位基因是纯合的，并且含有两个拷贝的G418抗性基因。该方案提供了获得纯合突变改变细胞的简单方法，即，双敲除，并且应该通常适用于其它基因和ES细胞以外的细胞系。该方法还应该能够产生其中多于一个基因的两个等位基因都被破坏的细胞系。这些突变细胞应该提供有用的工具，以确定特定基因在细胞培养中的作用。
We have developed a simple method for producing embryonic stem (ES) cell lines whereby both alleles have been inactivated by homologous recombination and which requires a single targeting construct. Four different ES cell lines were created that were heterozygous for genes encoding two guanine nucleotide-binding protein subunits, alpha(i2) and alpha(i3), T-cell receptor-alpha, and beta-cardiac myosin heavy chain. When these heterozygous cells were grown in high concentrations or G418, many of the surviving cells were homozygous for the targeted allele and contained two copies of the G418 resistance gene. This scheme provides an easy method for obtaining homozygous mutationally altered cells, i.e., double knockouts, and should be generally applicable to other genes and to cell lines other than ES cells. This method should also enable the production of cell lines in which more than one gene have had both alleles disrupted. These mutant cells should provide useful tools for defining the role of particular genes in cell culture.