利用Red/ET同源重组构建新型CCR5Δ32打靶载体

摘要

目的:目前应用TALENs或CRISPR-Cas基因编辑技术修饰CCR5基因以获得抗HIV功能的研究策略基本是破坏CCR5基因而不是制造CCR5Δ32这样天然存在的抗HIV基因型.为此,我们尝试通过细菌内同源重组技术构建一个新颖的CCR5Δ32的打靶载体,以便能够用于制备CCR5Δ32基因突变细胞系.方法:首先利用Red/ET同源重组系统将rpsl-neo片段插入细菌人工染色体(BAC)的CCR5基因中,随后再次利用同源重组将一个不含32 bp区域的非选择性52 bp片段替换该rpsl-neo片段,从而将BAC中的CCR5基因改造成CCR5Δ32基因型,最后将loxp-neo-loxp抗性基因插入CCR5基因的第2内含子区域,制备出含有抗性基因的CCR5Δ32打靶载体,该载体可在Cre酶作用下删除neo基因但只留下一个loxp在基因的内含子区.结果:rpsl-neo片段成功插入CCR5之Δ32区并为非选择性52 bp片段替换,loxp-neo-loxp成功插入内含子区.结论:通过Red/ET同源重组技术获得CCR5A 32-loxp-neo-loxp打靶载体.%Objective:Recently modification of CCR5 locus for anti-HIV-1 infection was performed by the genome editing methods like TELENs or CRISPR-Cas with the strategies of disruption of CCR5.To obtain the exact mutation of Δ32 deletion at the locus,we employed a novel strategy for developing CCR5Δ32 targeting vector based on Red/ET homologous recombination.Methods:Firstly,a rpsl-neo cassette flanked by two homology arms to CCR5 was amplified by PCR and then electroporated into E.coli DH10B with the BAC bearing CCR5.Through Red/ET homologous recombination,a 52 bp DNA sequences containing Δ32 in CCR5 was replaced by rpsl-neo.Subsequently,a non-selectable fragment was electroporated and replaced the rpsl-neo resulting in Δ32 deletion at CCR5.Furthermore,a loxp-neo-loxp cassette was also introduced into intron 2 within CCR5 through the same machnism,which allows for G418 selection for future targeting human cells and will be cut under the Cre protein only leaving one loxp at the intron region.Results:rpsl-neo cassette was successfully inserted into CCR5 on BAC and replaced by non-selectable fragment and at the same time a loxp-neo-loxp cassette was also introduced into intron 2 of CCR5.Conclusion:CCR5Δ32-loxp-neo-loxp targeting vector was accessed by Red/ET homologous recombination.