te Riele H, Maandag E R, Berns A
Division of Molecular Genetics, The Netherlands Cancer Institute, Amsterdam.
Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5128-32. doi: 10.1073/pnas.89.11.5128.
A vast amount of data suggests that homologous recombination in mammalian cells is relatively rare as compared to random integration, imposing the need for sophisticated selection protocols to enrich for cells in which homologous recombination has occurred. We here show that one of the key factors in efficient homologous recombination is the use of isogenic DNA to prepare the targeting vectors. Homologous recombination at the retinoblastoma susceptibility gene (Rb) in embryonic stem cells derived from mouse strain 129 was 20-fold more efficient with a 129-derived targeting construct than with a BALB/c-derived construct. The two constructs were identical, except for a number of base sequence divergences between 129 and BALB/c DNA, including base-pair substitutions, small deletions/insertions, and a polymorphic CA repeat. Transfection with an isogenic DNA construct, containing 17 kilobases of homology, yielded a targeting frequency of 78% (of a total of 20,000 drug-resistant colonies), without the use of an enrichment protocol for homologous recombination. This result indicates that, also in mammalian cells, homologous recombination rather than random integration can be the predominant event.
大量数据表明，与随机整合相比，哺乳动物细胞中的同源重组相对较少，这就需要复杂的筛选方案来富集发生同源重组的细胞。我们在此表明，高效同源重组的关键因素之一是使用同基因DNA制备靶向载体。在源自129小鼠品系的胚胎干细胞中，视网膜母细胞瘤易感基因（Rb）处的同源重组，使用源自129的靶向构建体比使用源自BALB/c的构建体效率高20倍。这两个构建体是相同的，只是129和BALB/c DNA之间存在一些碱基序列差异，包括碱基对替换、小的缺失/插入以及一个多态性CA重复序列。用含有17千碱基同源性的同基因DNA构建体转染，在不使用同源重组富集方案的情况下，靶向频率达到78%（在总共20000个抗药菌落中）。这一结果表明，在哺乳动物细胞中，同源重组而非随机整合也可能是主要事件。
