Construction of Chicken (Gallus gallus) Wnt5a Lentiviral Interference Vector and Screening of Stably Expressed Embryonic Stem Cell Lines
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
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pGMLV-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qRT-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qRT-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(pCDNA3.0-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-shRNA1、Wnt5a-shRNA2和Wnt5a-shRNA3,Wnt5a-shRNA2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-shRNA2重组质粒包裹慢病毒,病毒滴度：5×108 TU/mL;将Wnt5a-shRNA2慢病毒转染鸡ESCs后,qRT-PCR和Western blot结果表明,干扰组Wnt5a mRNA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shRNA慢病毒表达载体,可有效沉默Wnt5a基因在鸡DF-1细胞和胚胎干细胞中的表达,并且稳定表达Wnt5a-shRNA2的胚胎干细胞系可干扰Wnt信号转导,该结果将为进一步在细胞水平开展Wnt5a基因及其介导的Wnt信号通路的功能研究提供理论依据。
Abstract：Previous study had shown that Wnt family member 5A (Wnt5a) 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 Wnt5a-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 Wnt5a gene was established by Lentivirus mediated RNA interference technique. Three interference target sequences and one negative control sequence were designed for Wnt5a gene and recombined with the pGMLV-SC5 lentivirus vector. Chicken DF-1 cells were infected with lentivirus recombinant vector, and the expression of Wnt5a gene was detected by qRT-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 Wnt5a 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 qRT-PCR and Western blot. At the same time, a Wnt5a overexpression vector (pCDNA3.0-Wnt5a) was constructed to carry out the rescue experiment of Wnt5a gene. The recombinant plasmid of Wnt5a-shRNA1, Wnt5a-shRNA2 and Wnt5a-shRNA3 was successfully constructed, and the inhibition effect of the Wnt5a-shRNA2 was the best, and the interference efficiency was up to 80% (80%±0.02). The results of qRT-PCR showed the Wnt5a-shRNA2 recombinant plasmid was packaged into a retrovirus with a virus titer of 5×108 TU/mL. After transfecting Wnt5a-shRNA2 lentivirus into chicken ESCs, The results of qRT-PCR and Western blot showed that Wnt5a mRNA 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 levels of key genes Axin2 and β-catenin downstream of the Wnt signaling pathway were significantly down-regulated (0.41±0.08 vs 1.03±0.05; 0.25±0.04 vs 1.03±0.05)(P<0.05). At the same time, the Wnt5a overexpression vector (PCDNA3.0-Wnt5a) was transfected into the interference group, and the expression level of Wnt5a in the interference group was recovered (1.19±0.07 vs 1.03±0.02). In this study, a lentiviral vector expressing shRNA targeting chicken Wnt5a was successfully constructed, which could effectively silence the expression of Wnt5a gene in chicken DF-1 cells and ESCs. Moreover, ESCs lines stably expressing Wnt5a-shRNA2 could interfere with Wnt signaling transduction. These results will provide a theoretical basis for further research on the function of Wnt5a gene and its mediated Wnt signaling pathway at the cellular level.
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