同时进行Cre催化的两个等位基因重组，以恢复新霉素敏感性并促进纯合突变。
Simultaneous Cre catalyzed recombination of two alleles to restore neomycin sensitivity and facilitate homozygous mutations.
作者信息
Milstone D S, Bradwin G, Mortensen R M
机构信息
Vascular Research Division, Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
出版信息
Nucleic Acids Res. 1999 Aug 1;27(15):e10. doi: 10.1093/nar/27.15.e10.
Cells homozygous for neo-expressing mutations can be derived by culturing heterozygotes with elevated G418. We demonstrate that this strategy is significantly less efficient if hyg is substituted for neo. Therefore, to introduce additional mutations Cre recombinase was used to remove floxed neo from both alleles of homozygotes at two different loci. The rate-determining step in Cre excision appeared independent of substrate copy number. Incorporating cytosine deaminase and Herpes simplex virus thymidine kinase allowed negative selection for both targeting and Cre excision. The resulting G418-sensitive homozygous mutants should allow mutagenesis at additional loci and avoid untoward effects of retained selection markers.
通过用高浓度的G418培养杂合子，可以获得携带新霉素表达突变的纯合细胞。我们证明，如果用潮霉素替代新霉素，这种策略的效率会显著降低。因此，为了引入额外的突变，使用Cre重组酶从两个不同位点的纯合子的两个等位基因中去除loxP侧翼的新霉素。Cre切除的限速步骤似乎与底物拷贝数无关。引入胞嘧啶脱氨酶和单纯疱疹病毒胸苷激酶允许对靶向和Cre切除进行阴性选择。由此产生的对G418敏感的纯合突变体应该允许在其他位点进行诱变，并避免保留的选择标记带来的不良影响。
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