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Issue:ISSN 1000-7083
          CN 51-1193/Q
Director:Sichuan Association for Science and Technology
Sponsored by:Sichuan Society of Zoologists; Chengdu Giant Panda Breeding Research Foundation; Sichuan Association of Wildlife Conservation; Sichuan University
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Your Position :Home->Past Journals Catalog->2015 Vol.34 No.3

The Study on Application of Bicistronic Vector in Transgenic Zebrafish Based on 2A Peptide
Author of the article:ZHANG Li1, LIU Chao1, ZHOU Xin2, XIE Ying1, LIU Shufeng1*
Author's Workplace:1. Hebei Key Lab of Laboratory Animal, Hebei Medical University, Shijiazhuang 050017, China;
2. Department of Medical Genetics, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430032, China
Key Words:2A peptide; zebrafish; bicistronic
Abstract:Objective To apply 2A peptide in generating dicistronic or polycistronic transgenic zebrafish. Methods GFP and Cherry were constructed to the Tol2 plasmid backbone; B cell stimulating factor (BAFF) was also inserted into the upstream or downstream of 2A sequence to analysis the position impact on transmembrane fusion protein expression, and to explore the multi-gene co-expression of transgenic zebrafish. GFP-2A-Cherry sequence was cloned into the Tol2 plasmid by InFusion method to obtain pTol-GFP-2A-Cherry plasmid, and this plasmid was then transfected into HeLa cells and microinjected into 1-cell stage fertilized embryos of zebrafish; fluorescence microscopy was used to trace the GFP and Cherry expression of HeLa cells in vitro and juvenile in vivo. the expression of GFP and Cherry protein was validated by Western blot; BAFF, as a candidate gene, was constructed as pTol2-GFP-2A-BAFF and pTol2-BAFF-2A-Cherry, and were then injected into 1-cell stage fertilized embryos followed by Western blot to determine the 2A's position effects on protein expression. Methods GFP and Cherry would individually express and showed consistently temporal expression; GFP-2A-Cherry fusion protein could be spliced into GFP and Cherry proportionally. The pTol2-GFP-2A-Cherry plasmid which was microinjected into 1-cell stage fertilized embryos could express GFP and Cherry protein alone; Both pTol2-GFP-2A-BAFF and pTol2-BAFF-2A-Cherry could express fusion protein in zebrafish, however, BAFF that located downstream of 2A peptide was easily cleavaged from the fusion protein. Conclusion 2A peptide building strategy can be carried out in zebrafish with single carrier and single promoters, therefore, it was proved that the use of 2A peptide strategy could achieve multi-gene expression. And it was also found that the localization of 2A peptide could directly affect the protein function.
2015,34(3): 338-344 收稿日期:2014-9-19
DOI:10.3969/j.issn.1000-7083.2015.03.003
分类号:Q78
基金项目:河北省科技条件建设项目(12965519D)
作者简介:张力(1984—),博士,讲师,研究方向:基因修饰疾病模型及相关机制研究
*通讯作者:刘树锋,副主任技师,研究方向:实验动物与疾病模型的标准化,E-mail:liushf@126.com
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