CRISPR/Cas9技术敲除人前列腺癌PC-3细胞BLM解旋酶基因的研究  

Research of CRISPR/Cas9 Technique to Knock Out Bloom Helicase Gene in Human Prostate Cancer PC-3 Cells

作　　者：陈琨[1,2,3] 许厚强 赵佳福[1,2] 段志强[1] 吴萍[1] Chen Kun;Xu Houqiang;Zhao Jiafu;Duan Zhiqiang;Wu Ping(Key Laboratory of Animal Genetics,Breeding and Production in the Pleteau Mountains Region,Ministry of Education,Guizhou University,Guiyang 550025,China;College of Life Science,Guizhou University,Guiyang 550025,China;Guizhou Province People＇s Hospital,Guiyang 550003,China)

机构地区：[1]贵州大学高原山地动物遗传育种与繁殖教育部重点实验室,贵阳550025 [2]贵州大学生命科学学院,贵阳550025 [3]贵州省人民医院,贵阳550003

出　　处：《中国细胞生物学学报》2018年第9期1547-1553,共7页Chinese Journal of Cell Biology

基　　金：国家自然科学基金(批准号:31360215)资助的课题~~

摘　　要：BLM解旋酶是人类RecQ解旋酶家族的重要一员,在DNA的复制、修复、重组、转录、端粒的维持等细胞代谢过程中具有重要的作用。BLM基因与前列腺癌易感性相关,可作为治疗前列腺癌的候选基因。该研究设计了5个Cas9/SgRNA载体,并通过T7E1酶筛选出活性最强的SgRNA3载体,利用脂质体LTX将其与供体载体共同导入前列腺癌PC-3细胞。BLa和Puro抗性筛选及荧光蛋白标记甄别获得带阳性标记的细胞。Western blot及RTq-PCR检测证明,前列腺癌PC-3细胞中的BLM解旋酶基因被成功敲除,为深入研究BLM解旋酶基因在前列腺癌中的作用提供了重要的科学数据。BLM helicase is an important member of the human Rec Q helicase family, which plays a crucial role in DNA replication, repair, recombinant, transcription, telomere maintenance and other cell metabolism. The BLM helicase gene is associated with susceptibility to prostate cancer and it is also a candidate gene for treatment of prostate cancer. In this study, the five Cas9/SgRNA vectors were designed, and we selected the most active SgRNA3 vector by using T7E1 enzyme. Two vectors of Cas9/SgRNA3 and donor vector were jointly transduced into human prostate cancer PC-3 cell by LTX liposomes. BLa and Puro resistance screenings and fluorescent protein markers were used to identify cells with positive markers. The BLM helicase gene in PC-3 cells of prostate cancer was successfully knocked out detected by Western blot and RTq-PCR tests. This study can provide the important scientific data which play a role of the BLM helicase in prostate cancer.

关 键 词：BLM解旋酶基因 CRISPR/Cas9表达载体 基因敲除 前列腺癌PC-3细胞 

分 类 号：R737.25[医药卫生—肿瘤]