Kdm2b基因敲入双loxp序列的NIH3T3细胞株的构建  

Construction of NIH3T3 cell line with double loxp sequence knocking into Kdm2b gene

作　　者：陈素珠 卢文显 林典梁[1] 杜生荣[1] 林运鸿[1] 郑备红[1] CHEN Suzhu;LU Wenxian;LIN Dianliang;DU Shengrong;LIN Yunhong;ZHENG Beihong(Fujian Maternity and Child Health Hospital,the Affiliated Hospital of Fujian Medical University,Fuzhou 350001,China;不详)

机构地区：[1]福建省妇幼保健院福建医科大学附属医院,福州350001 [2]福建医科大学转化医学研究院

出　　处：《山东医药》2020年第32期22-26,共5页Shandong Medical Journal

基　　金：福建省自然科学基金资助项目(2018J01233);国家重点研发计划(2018YFC1002105);福建省妇幼保健院院内课题(妇保院研17-29)。

摘　　要：目的利用CRISPR/Cas9技术与同源重组技术在组蛋白去甲基化酶Kdm2b基因的特定位置敲入双loxp序列,为后期制备条件性敲除鼠及基因功能研究提供基础。方法利用CRISPR/Cas9靶向性原理,根据Kdm2b基因第7个外显子的5臂端和第9个外显子的3臂端设计两个sgRNA,构建sgRNA-PX459重组质粒。从Kdm2b基因上克隆出同源臂,将同源臂插入带有loxp序列的Vector 5中,利用引物从Vector 5质粒克隆得到loxp-同源臂序列的双链Donor DNA。通过脂质体转染将sgRNA-PX459重组质粒和双链Donor DNA导入小鼠NIH3T3细胞,利用嘌呤霉素筛选细胞,挑选阳性单克隆。提取NIH3T3细胞基因组进行PCR,对PCR产物酶切鉴定,同时对Kdm2b基因进行测序。结果电泳和测序结果显示sgRNA-PX459重组质粒构建成功,双链Donor DNA测序结果与设计相符合,成功在NIH3T3细胞Kdm2b基因上第7个外显子的5臂端和第9个外显子的3臂端各敲入一个loxp序列。结论获得Kdm2b基因敲入双loxp序列的NIH3T3细胞株。Objective To knock the loxp sequence into the demethylase Kdm2b gene at a specific position by using the CRISPR/Cas9 technique and homologous recombination technique,so as to make a preliminary exploration for the preparation of conditional knockout mice and the study of gene function.Methods Under the CRISPR/Cas9 targeting principle,two sgRNA were designed according to the 5′end of the 7th exon and the 3′end of the 9th exon of Kdm2b gene,and the sgRNA-px459 recombinant plasmid was constructed.The homologous arm was cloned from Kdm2b gene and inserted into Vector 5.The double stranded Donor DNA of loxp-homologous arm sequence was cloned from Vector 5 plasmid with primers.SgRNA-px459 recombinant plasmid and double stranded Donor DNA were transfected into NIH3T3 cells by liposome transfection,positive monoclones were screened out by drugs,and the genome was extracted for PCR.PCR products were identified by enzyme digestion,and Kdm2b gene was sequenced at the same time.Results The electrophoresis and sequencing results showed that sgRNA-px459 recombinant plasmid was successfully constructed,the results of double-stranded Donor DNA sequencing were consistent with the design,and we successfully knocked a loxp sequence into the 5′end of the 7th exon and the 3′end of the 9th exon on the demethylase Kdm2b gene,respectively.Conclusion The NIH3T3 cell line with double loxp sequence knocking into Kdm2b gene was successfully constructed.

关 键 词：组蛋白去甲基化酶 Kdm2b基因 条件性基因敲除 基因编辑 CRISPR/Cas9系统 Cre-loxp系统 

分 类 号：R394[医药卫生—医学遗传学]