摘要
根据已知的BLG序列设计引物,通过PCR技术克隆同源臂序列,5′端同源臂2 264bp,包括外显子1,3′端同源臂4 461bp,包括全部的外显子3、4、5、6、7,分别连入克隆载体pMD18-T Simple载体中并测序。然后以含有正负筛选标记基因的ploxpⅡ载体为基础,将5′端和3′端同源臂片段先后连入其中,进行酶切、PCR鉴定。将构建好的基因敲除载体转化组成型表达Cre重组酶的大肠杆菌BM25.8,验证Loxp位点的活性。结果表明,构建了奶山羊BLG基因第2外显子缺失的基因敲除载体pBLG2T,且pBLG2T载体中的正选neo基因可以被Cre重组酶去除。为获得BLG 1条等位基因缺失型细胞株以及培育高产优质奶山羊新品种奠定基础。
The primers were designed according to the known sequence of BLG,then the homologous arms were amplified by PCR.The 5′homologous arm is 2 264 bp,which included exon 1,the 3′ homologous arm is 4 461 bp,which included exon 3,exon 4,exon 5,exon 6,exon 7.After that,the homologous arms were cloned into pMD18-T Simple vector and sequenced respectively,then the 5′ homologous arm and 3′ homologous arm were subconed into the gene targeting plasmid ploxpⅡsuccessively,which contained the positive and negative selection gene.Subsequently,the gene knockout vector was identified by restriction endonuclease digestion and PCR.To verify the bioactivity of loxp locus,the constructed gene knockout vector was transformed into the BM25.8 expressing Cre recombinase.The result indicates that the diary goat BLG gene knockout vector pBLG2T with the deletion of exon2 was sucessfully constructed,and the positive neo gene can be deleted by Cre recombinase.This laid the foundation for obtaining the BLG＋/-cell line and breeding new breed of dairy goat,which has the aspect of top quality and high yield.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2011年第6期858-863,共6页
Chinese Journal of Veterinary Science
基金
转基因生物新品种培育重大专项(2008ZX08008-004)
