鸡FGF8基因RNA干扰载体的构建及其对PGCs形成的影响  

Construction of RNA Interference Vector of Chicken(Gallus domesticus) FGF8 Gene and Its Effect on the Formation of PGCs

作　　者：王曼[1] 孙长花 李东[2] 李婷婷[1] 张文慧[1] 金晶[1] 左其生[1] 张亚妮[1] 陈国宏[1,3] 李碧春 WANG Man;SUN Chang-Hua;LI Dong;LI Ting-Ting;ZHANG Wen-Hui;JIN Jingl;ZUO Qi-Sheng;ZHANG Ya-Ni;CHEN Guo-Hong;LI Bi-Chun(College of Animal Science and Technology,Yang Zhou University,Yangzhou 225009,China;Reproductive Medicine Center,Nanjing Drum Tower Hospital Affiliated to Medical School of Nanjing University,Nanjing 210008,China;Institutes of Agricultural Science and Technology Development,Yangzhou University,Yangzhou 225009,China;Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]扬州大学动物科学与技术学院,扬州225009 [2]南京大学医学院附属鼓楼医院生殖医学中心,南京210008 [3]扬州大学农业科技发展研究院,扬州225009 [4]扬州大学教育部农业与农产品安全国际合作联合实验室,扬州225009

出　　处：《农业生物技术学报》2018年第9期1457-1466,共10页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31772582;No.31572390和No.31472087);江苏省现代农业初步研究计划(No.BE2015362)

摘　　要：原始生殖细胞(primordial germ cells,PGCs)作为精细胞的祖细胞,广泛用于研究精子的发生。成纤维细胞生长因子(fibroblast growth factor,FGF)家族对PGCs的形成至关重要。为了探究成纤维生长因子8(FGF8)的具体功能,本研究通过构建家鸡(Gallus domesticus)FGF8的短发夹RNA(short hairpin RNA,sh RNA)慢病毒干扰载体,获得稳定低表达FGF8的鸡胚胎干细胞株,并进一步研究FGF8在鸡胚胎干细胞(embryonic stem cells,ESCs)向PGCs分化中的功能。根据家鸡FGF8的转录本设计3个RNA干扰靶点,连接到p GMVL-SC5干扰载体,瞬时转染鸡胚成纤维细胞DF1。以q RT-PCR技术检测各靶点对FGF8的干扰效率,将干扰效率最佳的sh RNA载体进行慢病毒包装。在以慢病毒敲低FGF8的ESCs中进行视黄酸(retinoic acid,RA)诱导,观察各组细胞形态变化,收集不同时间的各组细胞,利用q RT-PCR、间接免疫荧光以及流式细胞术等方法,分析检测生殖相关标记基因的表达变化和PGCs形成效率。结果表明,FGF8慢病毒干扰载体构建成功,分别命名为sh FGF8-1、sh FGF8-2、sh FGF8-3,其中sh FGF8-2干扰效率最佳。将sh FGF8-2进行慢病毒包装,获得滴度为5×10^(8 )TU/m L、干扰效率为(70±4.31)%的慢病毒载体。对于FGF8表达抑制的ESCs,体外RA诱导4 d后,PGC样细胞显著增加,ESC标记基因Nanog表达极显著下调(P<0.01),PGC标记基因Cvh、C-kit表达极显著上调(P<0.01)。此外,免疫荧光及流式细胞术分析结果也进一步证实,诱导4 d后与对照组比较,FGF8低表达使CVH^+PGCs比例显著增加(P<0.01)。本研究成功构建了稳定转染鸡胚胎干细胞的FGF8特异性慢病毒载体,并证实FGF8在体外参与调控PGCs的形成。As early precursors for sperm cells, primordial germ cells （PGCs） are widely used to study spermatogenesis. Fibroblast growth factors （FGFs） are essential in regulating the formation of PGCs. In order to investigate the regulation of FGF8 on the formation of PGCs, we established a chicken embryonic stem cell line with FGF8 low expression, through constructing a small hairpin RNA （shRNA） lentivirus vector for chicken （Gallus domesticus） FGF8, and further investigate the role that FGF8 plays in the differentiation of male germ cells. Three shRNA interfering vectors targeting chicken FGF8 were constructed and transfected into chicken DF1 cells transiently. qRT-PCR was used to detect the interference efficiency of different interference targets on FGF8, and the shRNA vector with the best interference efficiency was packaged in lentivirus. The induction of retinoic acid （RA） was performed in an ES cell lines knocked down by FGF8, and the morphological changes of the cells in each group were observed, also, the cells of each group were collected on day 0, 2, 4 and 6, respectively. The expression of related reproductive genes, like Nanog, Oct4, Cvh, C-kit, Blimp1 and so on, and the efficiency of PGCs formation were detected by qRT-PCR, flow cytometry, and immunofluorescence. The results showed that 3 lentiviral interference vectors and 1 negative vector for FGF8 were successfully constructed and named as shFGF8-1, shFGF8-2, shFGF8-3, shNC. By transfecting DF1 cells, shFGF8-2 had been found the highest interference efficiency relative to shFGF8-1 and shFGF8-3. Hence, shFGF8-2 was packaged with lentivirus and the titer was 5×108 TU/mL, the interference efficiency was （70±4.31）% in ESCs. For ESCs with low expression of FGF8, we found that under normal RA-induced for 4 d, PGC-like cells were significantly increased, and the pluripotency associated gene Nanog, was significantly down regulated （P〈0.01）. Meanwhile, the germ cell marker gene Cvh, and C-kit, were significantly up-regulated （P〈

关 键 词：鸡 成纤维生长因子8基因(FGF8) 胚胎干细胞(ESC) 原始生殖细胞(PGC) 短发夹RNA(shRNA) 

分 类 号：S831.2[农业科学—畜牧学] Q254[农业科学—畜牧兽医]