鸡Wnt5a基因慢病毒干扰载体的构建及其稳定表达的胚胎干细胞系筛选  

Construction of Chicken(Gallus gallus) Wnt5a Lentiviral Interference Vector and Screening of Stably Expressed Embryonic Stem Cell Lines

作　　者：周静 金晶[1] 何娜娜[1] 张晨[1] 王曼[1] 左其生[1] 张亚妮[1] 李碧春[1] ZHOU Jing;JIN Jing;HE Na-Na;ZHANG Chen;WANG Man;ZUO Qi-Sheng;ZHANG Ya-Ni;LI Bi-Chun(College of Animal Science and Technology,Yangzhou University/Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province/Institutes of Agricultural Science and Technology Development/Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China,Yangzhou 225009,China)

机构地区：[1]扬州大学动物科学与技术学院/江苏省动物遗传繁育与分子设计重点实验室/农业科技发展研究院/教育部农业与农产品安全国际合作联合实验室

出　　处：《农业生物技术学报》2020年第2期260-269,共10页Journal of Agricultural Biotechnology

基　　金：国家重点研究发展计划(2017YFE0108000);国家自然科学基金(31772582; 31872341);江苏高校优势学科建设工程项目;扬州大学高层次人才支持计划项目(X20170702);江苏省高等学校大学生创新创业训练计划项目(201811117013Z)

摘　　要：本课题组前期通过高通量测序发现无翅型MMTV整合位点家族成员5A (winglesstype MMTV integration site family member 5A, Wnt5a)介导的Wnt/β-catenin信号在鸡(Gallus gallus)雄性生殖细胞形成过程中被显著富集,为研究Wnt5a基因介导的Wnt/β-catenin信号通路在鸡胚胎干细胞(embryonic stem cells, ESCs)向雄性生殖细胞分化过程中的功能,本研究通过慢病毒(Lentivirus)介导的RNA干扰技术,建立稳定干扰Wnt5a基因的鸡胚胎干细胞系。针对Wnt5a基因设计3个干扰靶点序列和1个阴性对照序列,与p GMLV-SC5慢病毒载体重组;将慢病毒重组载体分别转染鸡DF-1细胞,通过q RT-PCR检测Wnt5a基因的表达,以确定各干扰载体的干扰效率,并对干扰效果最好的重组载体进行慢病毒包裹。利用包裹干扰载体的慢病毒感染鸡ESCs,采用q RT-PCR及Western blot检测不同组别Wnt5a及Wnt信号通路下游关键基因轴抑制蛋白2(axis inhibition protein 2, Axin2)、β-链蛋白(beta catenin,β-catenin),磷酯酶C(phospholipase C, PLC)及泛素特异性肽酶54基因(ubiquitin specific peptidase 54, USP54)的表达量变化。同时构建Wnt5a过表达载体(p CDNA3.0-Wnt5a)进行Wnt5a基因的拯救实验。成功构建3条靶向Wnt5a的慢病毒干扰表达载体,分别为Wnt5a-sh RNA1、Wnt5a-sh RNA2和Wnt5a-sh RNA3,Wnt5a-sh RNA2重组质粒对Wnt5a基因的抑制效果最为明显,干扰效率可达80%,将Wnt5a-sh RNA2重组质粒包裹慢病毒,病毒滴度:5×108TU/m L;将Wnt5a-sh RNA2慢病毒转染鸡ESCs后,q RT-PCR和Western blot结果表明,干扰组Wnt5a m RNA表达和蛋白表达水平较对照组显著降低(0.16±0.03 vs 1.03±0.02)(P<0.05),Wnt信号通路下游关键基因Axin2和β-catenin的表达量发生了显著下调(0.41±0.08 vs 1.03±0.05;0.25±0.04 vs 1.03±0.05)(P<0.05);同时对干扰组转染Wnt5a过表达载体(PCDNA3.0-Wnt5a),结果发现干扰组Wnt5a的表达水平能够恢复(1.19±0.07 vs 1.03±0.02)。本研究成功构建了靶向鸡Wnt5a的sh RNA慢病毒Previous study had shown that Wnt family member 5 A(Wnt5 a) mediated Wnt/β-catenin signal was significantly enriched in the formation of chicken(Gallus gallus) male germ cells by high-throughput sequencing. To investigate the role of Wnt5 a-mediated Wnt/β-catenin signaling pathway in the differentiation of chicken embryonic stem cells(ESCs) into male germ cells, a chicken ESCs line stably interfering with Wnt5 a gene was established by Lentivirus mediated RNA interference technique. Three interference target sequences and one negative control sequence were designed for Wnt5 a gene and recombined with the p GMLVSC5 lentivirus vector. Chicken DF-1 cells were infected with lentivirus recombinant vector, and the expression of Wnt5 a gene was detected by q RT-PCR to determine the interference efficiency of each interference vector,and the recombinant vector with the best interference effect was wrapped with lentivirus. Chicken ESCs were infected with lentivirus wrapped with interference vector, and the expression levels of Wnt5 a and Wnt signaling pathways key genes: Axis inhibition protein 2(Axin2), Beta Catenin(β-catenin), phospholipase C(PLC) and ubiquitin specific peptidase 54(USP54) were detected by q RT-PCR and Western blot. At the same time, a Wnt5 a overexpression vector(p CDNA3.0-Wnt5 a) was constructed to carry out the rescue experiment of Wnt5 a gene. The recombinant plasmid of Wnt5 a-sh RNA1, Wnt5 a-sh RNA2 and Wnt5 a-sh RNA3 was successfully constructed, and the inhibition effect of the Wnt5 a-sh RNA2 was the best, and the interference efficiency was up to 80%(80% ± 0.02). The results of q RT-PCR showed the Wnt5 a-sh RNA2 recombinant plasmid was packaged into a retrovirus with a virus titer of 5×108 TU/m L. After transfecting Wnt5 a-sh RNA2 lentivirus into chicken ESCs, The results of q RT-PCR and Western blot showed that Wnt5 a m RNA and protein expression levels in the interference group were significantly lower than those in the control group(0.16±0.03 vs 1.03 ± 0.02)(P<0.05). The expression lev

关 键 词：无翅型MMTV整合位点家族成员5A(Wnt5a) shRNA 慢病毒载体 鸡胚胎干细胞 

分 类 号：S80[农业科学—畜牧兽医]