流动引物PCR高效筛选阳性XRCC1基因敲除小鼠细胞的方法

目的]探索流动引物PCR筛选阳性XRCC1基因敲除细胞的可行性。方法]构建基因敲除质粒,嘌呤霉素抗性基因5’端加上额外的来自野生型基因被敲除位点内的20 bp DNA序列。使用流动引物(floating primer)和这20 bp序列结合,PCR筛选阳性克隆。结果]利用流动引物成功筛选小鼠体细胞XRCC1(X射线修复交叉互补蛋白)一个等位基因敲除的阳性克隆子。结论]流动引物PCR方法可以应用于基因敲除小鼠体细胞的筛选。

Employing PCR with floating primer to effectively screen XRCC1 gene knock out mice cells

Objective] We attempted to utilize floating primer to screen XRCC1 gene knock out mice cells.Methods] A gene knock out vector was constructed,in which an additional 20 bp DNA sequence from the knocking-out sites of wild-type gene was added to the 5’ terminus of puromycin resistant gene.A primer named for floating primer was designed to anneal with this 20 bp DNA sequence.Results] By using floating primer,the mice cells that have one deleted XRCC1(X-ray repair cross complementing protein 1)allele were effectively screened.Conclusion] PCR with floating primer is a useful method for screening gene knock out mice cells.