基于CRISPR/Cas9技术构建UCA1基因敲除的肺腺癌顺铂耐药株A549/DDP细胞  

Construction of knock-out UCA1 gene of cisplatin resistant lung adenocarcinoma cell line A549/DDP based on CRISPR/Cas9 technology

作　　者：王君君[1] 章帆[1] 姜丰[1] 胡丽娟[1] 陈坚[1] 王瑜敏[1] WANG Jun-jun;ZHANG Fan;JIANG Feng;HU Li-juan;CHEN Jian;WANG Yu-min(Department of Laboratory Medicine,the First Affiliated Hospital of Wenzhou Medical University,Wenzhou,Zhejiang 325000,China)

机构地区：[1]温州医科大学附属第一医院医学检验中心,浙江温州325000

出　　处：《中国卫生检验杂志》2021年第21期2561-2565,共5页Chinese Journal of Health Laboratory Technology

基　　金：国家自然科学基金(81672088);浙江省自然基金(LY-19H200002);温州市科技局课题(Y20180548)。

摘　　要：目的利用CRISPR/Cas9技术构建lncRNA UCA1基因敲除的A549/DDP细胞株。方法设计并合成sgRNA引物序列,退火形成UCA1-sgRNA双链DNA,与线性化pGK1.1载体连接获得重组质粒;电转染至A549/DDP细胞,对pool细胞(混合克隆)测序检测敲除效率,提取pool的靶细胞进行PCR扩增获得杂交DNA;Cruiser^(TM)Enzyme酶切筛选阳性克隆细胞,并对阳性克隆进行测序、TA克隆测序、RT-PCR验证。结果重组质粒测序结果显示,4个UCA1敲除重组质粒在靶位点插入的片段位置、方向及序列与预期结果一致,证明4个UCA1敲除重组质粒均构建成功。克隆细胞酶切结果显示敲除条带明显小于对照条带;测序及TA克隆测序结果与酶切结果一致;RT-PCR显示UCA1表达水平明显低于对照组(P<0.001)。结论成功构建UCA1基因敲除的A549/DDP细胞株,为后续的进一步研究提供了实验基础。Objective To.construct knock-out IncRNA UCA1 gene of A549/DDP cell line based on CRISPR/Cas9 technolo­gy.Methods SgRNA primer sequences were designed and synthesized,and then annealed to form UC-A1-sgRNA double stranded DNA,which was related to the linearized pGK1.1 vector to obtain the recombinant plasmid.After transfection into A549/DDP cells,the pool cells(mixed clones)were sequenced to detect the knockout efficiency.The target cells of the pool were extracted for PCR amplification to obtain hybrid DNA.The positive clones were screened by Cruiser^(TM)Enzyme digestion,and the positive clones were verified by sequencing,Ta cloning sequencing and RT-PCR.Results The results of recombinant plasmid sequencing showed that the position,direction and sequence of the four UCA1 knockout recombinant plasmids were con­sistent with the expected results,which proved that the four UCA1 knockout recombinant plasmids were successfully construc­ted.The results of enzyme digestion showed that the knockout band was significantly smaller than the control band;sequencing and Ta cloning sequencing results were consistent with enzyme digestion results;RT-PCR showed that UCA1 level was signifi­cantly lower than that of control group(P<0.001).Conclusion The successful construction of knock-out UCA1 gene of A549/DDP cell line provides experimental basis for further research.

关 键 词：CRISPR/Cas9技术 长链非编码RNA A549/DDP细胞 UCA1基因 

分 类 号：R734.2[医药卫生—肿瘤]