HIGHLY EFFICIENT GENE TARGETING IN EMBRYONIC STEM-CELLS THROUGH HOMOLOGOUS RECOMBINATION WITH ISOGENIC DNA CONSTRUCTS
HIGHLY EFFICIENT GENE TARGETING IN EMBRYONIC STEM-CELLS THROUGH HOMOLOGOUS RECOMBINATION WITH ISOGENIC DNA CONSTRUCTS
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DOI：
10.1073/pnas.89.11.5128
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发表时间：
1992-06-01
影响因子：
11.1
通讯作者：
BERNS, A
中科院分区：
文献类型：
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作者：
RIELE, HT;MAANDAG, ER;BERNS, A
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罕见，这就需要复杂的选择方案来富集其中发生同源重组的细胞。我们在这里表明，在有效的同源重组的关键因素之一是使用同基因DNA制备的靶向载体。同源重组视网膜母细胞瘤易感基因（Rb）在胚胎干细胞来源于小鼠品系129是20倍更有效的129衍生的靶向构建体比BALB/c衍生的构建体。这两个构建体是相同的，除了129和BALB/c DNA之间的一些碱基序列的分歧，包括碱基对取代，小的缺失/插入，和多态性CA重复。用含有17个同源性内切酶的同基因DNA构建体转染，产生了78%的靶向频率（总共20，000个耐药菌落），而不使用用于同源重组的富集方案。这一结果表明，在哺乳动物细胞中，同源重组而不是随机整合可能是主要事件。
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 divergencies 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.