POSITIVE GENETIC SELECTION FOR GENE DISRUPTION IN MAMMALIAN-CELLS BY HOMOLOGOUS RECOMBINATION
POSITIVE GENETIC SELECTION FOR GENE DISRUPTION IN MAMMALIAN-CELLS BY HOMOLOGOUS RECOMBINATION
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DOI：
10.1073/pnas.86.1.227
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发表时间：
1989-01-01
影响因子：
11.1
通讯作者：
SHARP, PA
中科院分区：
文献类型：
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作者：
SEDIVY, JM;SHARP, PA
由于非同源重组的高频率，通过同源重组对哺乳动物细胞中的基因进行有效修饰是不可能的。一种有效的靶向基因破坏方法已经开发出来。使用特异性选择靶位点处同源重组的阳性遗传选择，将外源序列置换到染色体基因座中的细胞富集100倍。该选择基于显性选择标记的条件表达，其通过与靶基因的框内基因融合实现。显性选择标记是通过修饰大肠杆菌neo基因而得到的，使得其在与异源编码序列框内融合后在哺乳动物细胞中保留显著活性。在此处提供的示例中，从其中每10，000个掺入外源DNA的细胞中有1个靶基因被破坏的库中有效地回收了同源重组体。
Efficient modification of genes in mammalian cells by homologous recombination has not been possible because of the high frequency of nonhomologous recombination. An efficient method for targeted gene disruption has been developed. Cells with substitution of exogenous sequences into a chromosomal locus were enriched, by a factor of 100, using a positive genetic selection that specifically selects for homologous recombination at the targeted site. The selection is based on the conditional expression of a dominant selectable marker by virtue of in-frame gene fusion with the target gene. The dominant selectable marker was derived by modification of the Escherichia coli neo gene so that it retains significant activity in mammalian cells after in-frame fusion with heterologous coding sequences. In the example presented here, homologous recombinants were efficiently recovered from a pool in which the targeted gene was disrupted in 1 per 10,000 cells incorporating exogenous DNA.