Selection-marker-free modification of the murine ß-casein gene using a lox2722 site
Selection-marker-free modification of the murine ß-casein gene using a lox2722 site
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DOI：
10.1006/abio.2000.4984
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发表时间：
2001-03-15
影响因子：
2.9
通讯作者：
Kolb, AF
中科院分区：
文献类型：
--
作者：
Kolb, AF
基因靶向和位点特异性重组策略允许对真核基因组进行精确修饰。然而，目前使用的许多重组策略将在修饰位点引入选择标记基因。原核生物来源的DNA序列如载体序列、选择标记和报告基因已显示出显著影响修饰的基因组位点的调节。为了避免插入过量的序列，设计了涉及同源重组和Cre重组酶介导的盒交换（RMCE）的双相重组策略，并用于将外源基因插入鼠胚胎干细胞中的β-酪蛋白基因中。发现用于重组酶介导的盒交换的异源特异性Lox位点的不相容性对于该策略的成功至关重要。经常使用的突变位点lox 511，其不同于天然loxP位点的一个单点突变，证明不适合这种方法。然而，携带两个点突变的突变体lox位点是高度有效的，并且90%的所选细胞克隆携带所需的修饰。这种双相重组策略允许在不伴随引入选择性标记基因的情况下对基因座进行有效和精确的修饰。(C)北京：科学出版社.
Gene targeting and site-specific recombination strategies allow the precise modification of the eukaryotic genome. Many of the recombination strategies currently used, however, will introduce a selection marker gene at the modified site. DNA sequences of prokaryotic origin like vector sequences, selection marker, and reporter genes have been shown to markedly influence the regulation of the modified genomic loci. In order to avoid the insertion of excess sequences, a biphasic recombination strategy involving homologous recombination and Cre-recombinase-mediated cassette exchange (RMCE) was devised and used to insert a foreign gene into the beta -casein gene in murine embryonic stem cells. The incompatibility of the heterospecific Lox sites used for the recombinase-mediated cassette exchange was found to be critical for the success of the strategy. The frequently used mutant site lox511, which differs from the natural loxP site by a single point mutation, proved unsuitable for this approach. A mutant lox site carrying two point mutations, however, was highly effective and 90% of the selected cell clones carried the desired modification. This biphasic recombination strategy allows for the efficient and precise modification of gene loci without the concomitant introduction of a selectable marker gene. (C) 2001 Academic Press.