应用CRISPR/Cas9技术敲除小鼠海马神经元中Purα基因可减弱神经元对DNA损伤的修复作用  

Knock Out Purα Gene in Mouse Hippocampus Neuronal Cells with CRISPR/Cas9 Techniques Could Deteriorate Its DNA Repair Effects of the Neuron

作　　者：张冰莹 郭姗姗[1] 石晓光[1] 何文欣[1] 刘昆梅[1] 孙涛[1] 崔建奇[1,2] Zhang Bingying;Guo Shanshan;Shi Xiaoguang;He Wenxin;Liu Kunmei;Sun Tao;Cui Jianqi(Ningxia Key Laboratory of Cerebrocranial Diseases, Incubation Base of National Key Laboratory, Ningxia Medical University, Finchuan 750004, China;Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Ningxia Medical University, ~nehuan 750004, China)

机构地区：[1]宁夏医科大学宁夏颅脑疾病重点实验室省部共建国家重点实验室培育基地,银川750004 [2]宁夏医科大学基础医学院生物化学与分子生物学系,银川750004

出　　处：《中国细胞生物学学报》2018年第5期706-716,共11页Chinese Journal of Cell Biology

基　　金：国家自然科学基金(批准号:81260197);宁夏脑计划项目(批准号:2016BZ07);宁夏医科大学2015年优势学科群建设科研项目(批准号:XY201511)资助的课题~~

摘　　要：人转录活化因子Purα是体内重要的转录因子,在体内多个环节中都发挥着重要的作用,尤其是在神经系统中,它与神经元的发育,突触形成都有很密切的关系。该文通过构建Purα基因敲除的小鼠海马神经元细胞系来观察其在DNA损伤修复中的作用。根据目的基因靶向删除外显子的位置设计相应的sg RNA序列,合成相应的寡核苷酸后克隆在p Sp Cas9(BB)-2A-Puro(PX459)V2.0质粒载体相应位点上,从而构建能对Purα基因进行敲除的CRISPR/Cas9质粒,将构建好的质粒通过酶切和测序进行鉴定,将经过鉴定证实相位正确及在有sg RNA序列插入的阳性质粒转染到小鼠海马神经元(HT22)细胞中,加入嘌呤霉素进行抗性筛选,保留正常生长的细胞进行培养,从而建立能稳定地进行Purα基因敲除的细胞系。通过Real-time PCR和Western blot技术分别在转录和翻译水平上检测Purα基因的表达情况,从而来判定Purα基因的敲除效率;将所构建的Purα基因敲除的细胞用羟基脲(HU)进行处理来建立细胞DNA损伤的实验模型,通过Western blot、脉冲电场反转凝胶电泳实验及细胞毒性实验观察Purα在DNA损伤修复中的作用。实验结果证实,所插入片段相位正确。通过TA克隆测序和T7E1酶切证实所构建的质粒具有良好的CRISPR/Cas9活性,可导致基因组碱基发生突变,Real-time PCR和Western blot实验结果证实,Purα基因敲除组的Purα基因表达明显降低。Purα基因敲除后,细胞对HU引起的DNA损伤极为敏感,说明Purα在维持基因组DNA的完整性方面发挥着重要的作用,是细胞中一种不可或缺的蛋白质。该实验结果还提示,利用CRISPR/Cs9技术可以有效地对目的基因进行敲除,同时由于该质粒携带有嘌呤霉素抗性基因,利用这一特点,可以方便地建立稳定的细胞系,该细胞系可用作研究Purα基因在神经元中的生物学功能的细胞模型。Human transcriptional activator Purα is an important transcriptional factor in the body and plays important roles in many aspects, especially in the nervous system. Purα is closed associated with the neuronal development and the formation of the synaptic plasticity. With establishment of stable Purα gene knock-out mouse hippocampus neuronal cell lines, the current study observed the effects of Purα on damaged DNA repair. According to the exon position in the targeted gene, the oligonucleotides with corresponding sg RNA sequences were designed, and the synthesized oligonucleotides were cloned into the corresponding cloning sites of the p Sp Cas9（BB）-2 APuro（PX459） V2.0 plasmid so that Purα gene knocked out plasmids were established. The positive clones were selected and identified with endonuclease cutting and sequencing analysis and then the plasmids with correct ORF and sg RNA insert were transferred into mouse hippocampus neuronal cells（HT22）, and selected with purimycin, the survived cells were kept for the further cultivation and the stable cell lines with Purα gene knocked out were established. The Real-time PCR and Western blot analysis were employed to check the expression levels of Purα gene in transcriptional and translational levels respectively so that the efficiency of Purα gene knock out were evaluated.The established cell lines were treated with HU to build the experimental model of DNA damage. The effects of Purα on DNA damage were observed with Western blot assay, pulse field reverse electrophoresis as well as the cellular proliferation and cytotoxicity test analysis. The experimental results demonstrated that the inserted fragment with correct ORF. The TA cloning sequences and T7 E1 endonuclease cutting proved that the constructed plasmids possesses the ideal and effective CRISPR/Cas9 activities and could cause the base mutation of the genomic DNA, the results of Real-time PCR and Western blot assay confirmed that Purα gene expression decreased remarkably in the gene knock o

关 键 词：CRISPR/Cas9 Purα 基因敲除 羟基脲 细胞损伤修复 

分 类 号：Q78[生物学—分子生物学] R741[医药卫生—神经病学与精神病学]