Christ Nicole, Dröge Peter
Institute of Genetics, University of Cologne, Cologne, Germany.
Genesis. 2002 Mar;32(3):203-8. doi: 10.1002/gene.10031.
Mutant lambda integrases catalyze site-specific recombination reactions inside mammalian cells. Here we demonstrate that the integrase system can be used to eliminate resistance marker genes from the genome of mouse embryonic stem cells. So-called integrative and excisive recombination pathways led to the precise deletion of the neomycin gene, which was inserted together with a flanking pair of directly repeated recombination sites into the ROSA26 locus by standard targeting techniques. The excision of the resistance gene led to the expression of enhanced green fluorescence protein, which served as a means to sort out cells that had undergone site-specific recombination. Southern analysis and DNA sequencing confirmed that strand exchange reactions had occurred in the genome as expected. Hence, the integrase system may be used in conjunction with other site-specific recombinases as a tool in genome manipulation protocols.
突变型λ整合酶可在哺乳动物细胞内催化位点特异性重组反应。在此，我们证明整合酶系统可用于从小鼠胚胎干细胞基因组中消除抗性标记基因。所谓的整合和切除重组途径导致新霉素基因的精确缺失，该基因通过标准靶向技术与侧翼的一对直接重复重组位点一起插入到ROSA26基因座中。抗性基因的切除导致增强型绿色荧光蛋白的表达，该蛋白作为一种筛选出经历了位点特异性重组的细胞的手段。Southern分析和DNA测序证实基因组中已按预期发生了链交换反应。因此，整合酶系统可与其他位点特异性重组酶结合使用，作为基因组操作方案中的一种工具。
