Department of Human Genome Research, Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba, Japan.
Biotechniques. 2011 Apr;50(4):235-46. doi: 10.2144/000113649.
Site-specific recombination is a powerful biotechnological tool for genome engineering. We previously reported two novel site-specific recombination systems, VCre/VloxP and SCre/SloxP, that do not cross-react with Cre/loxP and Flp/FRT in culture cells and mouse embryonic stem (ES) cells. In this study, a site-specific recombination assay in Escherichia coli was used to examine the activity of mutant VCre (H314L and Y349F) and mutant SCre (H317L and Y352F), in which both mutated residues lie within the active center of Cre recombination. The site-specific recombination activity of both mutants was significantly decreased. Recombinase-mediated cassette exchange (RMCE) using VloxP and the Vlox2272 mutant site was performed in E. coli by introducing a cassette bearing VloxP and Vlox2272 into a recipient plasmid bearing the same sites. RMCE using SloxP and Slox2272 was also performed by SCre recombinase. Moreover, BAC engineering via Red recombination and VCre/VloxP were demonstrated. First, the DNA cassette for modification was introduced into a BAC clone via Red recombination; second, the antibiotics resistance gene flanked by VloxP was removed from the BAC clone by induction of VCre recombinase. Such site-specific recombination systems may effectively be used in combination with other site-specific recombination systems or engineering tools (e.g., Red recombination).
位点特异性重组是用于基因组工程的强大生物技术工具。我们之前报道了两种新型的位点特异性重组系统，VCre/VloxP 和 SCre/SloxP，它们在培养细胞和小鼠胚胎干细胞（ES 细胞）中与 Cre/loxP 和 Flp/FRT 不发生交叉反应。在这项研究中，我们使用大肠杆菌中的位点特异性重组测定来研究突变 VCre（H314L 和 Y349F）和突变 SCre（H317L 和 Y352F）的活性，这两种突变残基都位于 Cre 重组的活性中心内。两种突变体的位点特异性重组活性均显著降低。通过将带有 VloxP 和 Vlox2272 突变位点的基因盒引入携带相同位点的受体质粒中，在大肠杆菌中进行 VloxP 和 Vlox2272 突变位点的重组酶介导的盒交换（RMCE）。通过 SCre 重组酶也进行了 SloxP 和 Slox2272 的 RMCE。此外，还通过 Red 重组和 VCre/VloxP 进行了 BAC 工程。首先，通过 Red 重组将用于修饰的 DNA 盒引入 BAC 克隆中；其次，通过诱导 VCre 重组酶去除 BAC 克隆中由 VloxP 侧翼的抗生素抗性基因。这些位点特异性重组系统可以与其他位点特异性重组系统或工程工具（例如，Red 重组）有效地结合使用。
