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| 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 |
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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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Received: 27 June 2019
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Corresponding Authors:
*yubcli@yzu.edu.cn
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[1] 蒋玉玲, 刘波, 丛蔚, 等. 2017. Wnt5a基因下调对C212细胞增殖与分化的影响[EB/OL]. 北京: 中国科技论文在线[2017-04-26].
(Jiang Y Q, Liu B, Cong W, et al.2017. Down-regulation of wnt5a gene affects proliferation and differentiation of c212 myogeneic cells. Chinese scientific papers online [2017-04-26].)
[2] 李凤立, 靳卫国. 2013. Wnt蛋白分泌机制的研究进展[J]. 泰山医学院学报, 34(11): 884-886.
(Li F L, Jin W G.2013. Advances in the study of wnt protein secretion mechanisms[J]. Journal of Taishan Medical College, 34(11): 884-886.)
[3] 孟凡荣, 陈琛, 万海粟, 等. 2014. 慢病毒载体及其研究进展[J]. 中国肺癌杂志, (12): 870-876.
(Meng F R, Chen C, Wan H L, et al. 2014. Advances of lentiviral vectors[J]. Chinese Journal of Lung Cancer, (12): 870-876.)
[4] 张世蘋, 张旭. 2017. Wnt信号通路在肿瘤调控方面的研究进展[J]. 中国药理学通报, 33(1): 14-18.
(Zhang S P, Zhang X.2017. Research progress on role of Wnt signaling pathway in regulation of tumors[J]. Chinese Pharmacological Bulletin, 33(1): 14-18.)
[5] 甄辉, 吴素格, 鹿晴晴,等. 2016. 东亚三角涡虫DjWnt5a克隆及在胚胎不同发育阶段中的表达[J]. 中国生物化学与分子生物学报, 32(2): 199-206.
(Zhen H, Wu S G, Lu Q Q, et al.2016. Cloning and expression of the dj wnt5a gene in planarian Dugesia japonica during different embryonic development stages[J]. Chinese Journal of Biochemistry Molecular Biology, 32(2): 199-206.)
[6] Alok A, Lei Z, Jagannathan N S, et al.Wnt proteins synergize to activate β-catenin signaling[J]. Journal of Cell Science, 2017, 130(9):1532-1544.
[7] Chawengsaksophak K, Svingen T, Ng E T, et al.Loss of Wnt5a disrupts primordial germ cell migration and male sexual development in mice1[J]. 2012, 86(1):1-12.
[8] Fan J, Wei Q, Liao J, et al.2017. Noncanonical Wnt signaling plays an important role in modulating canonical Wnt-regulated stemness, proliferation and terminal differentiation of hepatic progenitors[J]. Oncotarget, 8(16): 27105-27119.
[9] He N, Wang Y, Zhang C, et al.2017. Wnt signaling pathway regulates differentiation of chicken embryonic stem cells into spermatogonial stem cells via wnt5a[J]. Journal of Cellular Biochemistry, 119(2).
[10] Kai K, Clevers H.2017. Wnt/β-catenin signaling in adult mammalian epithelial stem cells[J]. Developmental Biology, 428(2): 273-282.
[11] Logan C Y, Nusse R.2004. The wnt signaling pathway in development and disease[J]. Annual Review of Cell and Developmental Biology, 20(1): 781-810.
[12] Mills K M, Szczerkowski J L A, Habib S J .2017. Wnt ligand presentation and reception: From the stem cell niche to tissue engineering[J]. Open Biology, 7(8):170140.
[13] Muruganandan S, Govindarajan R, Mcmullen N M, et al.2017. Chemokine-Like Receptor 1 Is a Novel Wnt Target Gene that Regulates Mesenchymal Stem Cell Differentiation[J]. STEM CELLS, 35(3):711-724.
[14] Reya T, Clevers H.2005. Wnt signalling in stem cells and cancer[J]. Nature, 434: 843-50.
[15] Schambony A, Wedlich D.2007. Wnt-5A/Ror2 regulate expression of XPAPC through an alternative noncanonical signaling pathway[J]. Developmental Cell, 12(5): 779-792.
[16] Tanaka T, Kanatsu-Shinohara M, Lei Z, et al.2016. The luteinizing hormone-testosterone pathway regulates mouse spermatogonial stem cell self-renewal by suppressing wnt5a expression in sertoli cells[J]. Stem Cell Reports, 7(2): 279-291.
[17] Toshio Miki, Shin-ya Yasuda, Michael Kahn.2011. Wnt/β-catenin signaling in embryonic stem cell self-renewal and somatic cell reprogramming[J]. Stem Cell Reviews & Reports, 7(4): 836-846.
[18] Yang J, Zhang K, Wu J, et al.2016. Wnt5a increases properties of lung cancer stem cells and resistance to cisplatin through activation of wnt5a/pkc signaling pathway[J]. Stem Cells International, 2016(1): 1-16.
[19] Yeh J R, Zhang X, Nagano M C.2011. Wnt5a is a cell-extrinsic factor that supports self-renewal of mouse spermatogonial stem cells[J]. Journal of Cell Science, 124(14): 2357-2366.
[20] Zhang Y, Tu C, Zhang D, et al.2015. Wnt/β-Catenin and Wnt5a/Ca2+ Pathways Regulate Proliferation and Apoptosis of keratinocytes in psoriasis lesions[J]. Cellular Physiology and Biochemistry, 36(5):1890-1902. |
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