人结直肠癌细胞Drosha基因敲除细胞模型的构建

目的 为研究核酸酶Drosha与结直肠癌的关系,利用腺相关病毒（AAV）介导的染色体同源重组技术,构建Drosha基因敲除的HCT116结直肠癌细胞模型.方法 设计引物通过PCR扩增Drosha基因的同源臂,构建Drosha的AAV病毒打靶载体,通过病毒的包装、感染、抗生素新霉素（G418）药物筛选、PCR以及Western blot鉴定获得基因敲除阳性细胞株,通过Cre病毒感染去除抗性基因,最终建立敲除Drosha基因的结直肠癌细胞模型.结果 经过两轮打靶,分别将DNA双链上的Drosha基因去除并经Western blot鉴定,获得Drosha-/-阳性的HCT116细胞株.结论 通过AAV病毒介导的染色体同源重组技术,成功构建Drosha基因敲除的HCT116细胞系.

Establishment of Drosha-/-cell Model in HCT116

Objective To investigate the role of Drosha involved in colorectal tumorgenesis, the AAV - mediated homologous recom bination technology was developed to construct a human colorectal cell in which Drosha is completely deleted. Methods The homologous arms for AAV -targeted deletion of Drosha were amplified by PCR from HCTll6 genomic DNA and cloned into the AAV expression vectors, which were packaged into 293T cells to produce the live viruses. HCTll6 cells were infected by the viruses and the positive cells were identified by G418 selection, PCR screening and Western blot. Finally, the neomycin gene in the identified ceils was removed by the Cre virus. Results Drosha -/-positive cell clones were successfully constructed after Co418 selection, PCR screening, Western blot and Cre vi rus infection. Conclusion The HCT116 Drosha-/-cell was successfully established by AAV -mediated deletion technology.