p53^(V149F)突变打靶载体及细胞模型的构建  

Construction of p53^(V149F) Mutant Target Carrier and Cell Model

作　　者：曹攀 蔡洁 张晓银 熊喆 角德灵 赵恒 徐凯祥 赵红业 CAO Pan;CAI Jie;ZHANG Xiaoyin;XIONG Zhe;JIAO Deling;ZHAO Heng;XU Kaixiang;ZHAO Hongye(Key Laboratory of Animal Gene Editing and Cell Cloning in Yunnan Province,Kunming 650201,China;Xenotransplantation Research Engineering Center in Yunnan Province,Kunming 650201,China;College of Plant Protection,Yunnan Agricultural University,Kunming 650201,China;College of Animal Medicine,Yunnan Agricultural University,Kunming 650201,China)

机构地区：[1]云南省动物基因编辑与体细胞克隆技术重点实验室,云南昆明650201 [2]云南省异种器官移植工程研究中心,云南昆明650201 [3]云南农业大学植物保护学院,云南昆明650201 [4]云南农业大学动物医学院,云南昆明650201

出　　处：《云南农业大学学报（自然科学版）》2024年第3期64-71,共8页Journal of Yunnan Agricultural University:Natural Science

基　　金：云南省重大科技专项计划项目(202102AA100054)。

摘　　要：[目的]构建p53基因突变模型,为研究p53基因突变引发肿瘤的发生和进展提供细胞模型资源。[方法]利用CRISPR/Cas9基因编辑技术,根据p53^(V149F)突变位点的序列,在线设计合成单链向导sgRNA,构建CRISPR打靶载体;将重组载体转染细胞,流式分选阳性细胞群,提取细胞基因组,Sanger测序分析Cas9核酸酶对靶位点的切割情况,构建同源修复模板,通过T7EN1试验进一步检测编辑效率;将重组载体和同源模板通过电转染方法共同转染至成纤维细胞中,通过单克隆挑取、PCR和Sanger测序鉴定挑取含目标突变的细胞株。[结果]成功构建靶向p53基因目标区域的CRISPR/Cas9打靶载体;获得5个含目标突变的克隆点,点突变率为10%,测序结果出现套峰,表明出现基因型多样化;1个细胞克隆的测序结果无杂峰,TA克隆鉴定结果表明:一个等位基因存在p53^(V149F)位点突变,另一个等位基因存在240 bp缺失。[结论]成功构建了含p53^(V149F)的细胞系,并对基因的功能进行了初步验证,为进一步构建两基因同时修饰模型奠定了基础,并为药物筛选提供可用的细胞模型资源,为创制模拟疾病发生的遗传模式和临床表现的小型猪实验动物模型奠定了基础。[Purpose] To construct p53 gene mutation model,providing cell model resources for studying the occurrence and progression of tumor induced by p53 gene mutation.[Methods] According to the sequence of p53^(V149F) mutation site,CRISPR/Cas9 gene editing technology was used to design and synthesize the single-strand wizard sgRNA online,and construct CRISPR target vector.The recombinant vector was transfected into cells,positive cell populations were sorted by flow cytometry,and cell genome was extracted.Sanger sequencing was conducted to analyze the cleavage of target sites by Cas9 nuclease,and homologous repair template was constructed.The editing efficiency was further detected by T7EN1 assay.The recombinant vector and homologous template were transfected into fibroblasts by electrotransfection,and cell lines containing target mutations were selected by monoclonal selection,PCR and Sanger sequencing.[Results] The CRISPR/Cas9 vector targeting p53 gene was successfully constructed.Five clone sites with target mutation were obtained,and the mutation rate was 10%.There were jacking peaks in the sequencing results,indicating that the genotypes were diverse.The sequencing results of one cell clone showed no heteropeak,and the genotype was identified by TA clone.The results showed that one allele had p53^(V149F) mutation,and the other allele had 240 bp deletion.[Conclusion] The cell line containing p53^(V149F) is constructed successfully and the function of the gene is preliminarily verified.It lays a preliminary foundation for the further construction of the model of the simultaneous modification of two genes,providing available cell model resources for drug screening,and laying a foundation for the creation of miniature pig experimental animal models that simulate the genetic pattern and clinical manifestations of disease occurrence.

关 键 词：基因编辑 打靶载体 癌症 P53基因 

分 类 号：Q78[生物学—分子生物学]