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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
Address:College of Life Sciences, Sichuan University, No.29, Wangjiang Road, Chengdu, Sichuan Province, 610064, China
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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
谢英, 梁卫华, 王冀, 等. 2013. 一种低成本实验室用斑马鱼饲养繁育设备[J]. 科学技术与工程, 13(14): 150-155.
Berghmans S, Jette C, Langenau D, et al. 2005. Making waves in cancer research: new models in the zebrafish[J]. Biotechniques, 39(2): 227-237.
Chan HY, Sivakamasundari V, Xing X, et al. 2011. Comparison of IRES and F2A-based locus-specific multicistronic expression in stable mouse lines[J]. PLoS One, 6(12): e28885.
de Felipe P, Hughes LE, Ryan MD, et al. 2003. Co-translational, intraribosomal cleavage of polypeptides by the foot-and-mouth disease virus 2A peptide[J]. J Biol Chem, 278(13): 11441-11448.
de Felipe P. 2002. Polycistronic viral vectors[J]. Curr Gene Ther, 2(3): 355-378.
Deng W, Yang D, Zhao B, et al. 2011. Use of the 2A peptide for generation of multi-transgenic pigs through a single round of nuclear transfer[J]. PLoS One, 6(5): e19986.
Ibrahimi A, Vande Velde G, Reumers V, et al. 2009. Highly efficient multicistroniclentiviral vectors with peptide 2A sequences[J]. Hum Gene Ther, 20(8): 845-860.
Kawakami K. 2005. Transposon tools and methods in zebrafish[J]. Dev Dyn, 234(2): 244-254.
Kim JH, Lee SR, Li LH, et al. 2011. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice[J]. PLoS One, 6(4): e18556.
Lin YJ, Huang LH, Huang CT. 2013. Enhancement of heterologous gene expression in Flammulina velutipes using polycistronic vectors containing a viral 2A cleavage sequence[J]. PLoS One, 8(3): e59099.
Rothwell DG, Crossley R, Bridgeman JS, et al. 2010. Functional expression of secreted proteins from a bicistronic retroviral cassette based on foot-and-mouth disease virus 2A can be position dependent[J]. Hum Gene Ther, 21(11): 1631-1637.
Schartl M. 2014. Beyond the zebrafish: diverse fish species for modeling human disease[J]. Dis Model Mech, 7(2): 181-192.
TianY, Li W, Wang L, et al. 2013. Expression of 2A peptide mediated tri-fluorescent protein genes were regulated by epigenetics in transgenic sheep[J]. Biochem Biophys Res Commun, 434(3): 681-687.
Ward KA. 1991. The application of transgenic techniques for the improvement of domestic animal productivity[J]. Curr Opin Biotechnol, 2(6): 834-839.
Yen HH, Scheerlinck JP. 2013. Biological activity of ovine IL-23 expressed using a foot-and-mouth disease virus 2A self-cleaving peptide[J]. Cytokine, 61(3): 744-746.
Zon LI. 1999. Zebrafish: a new model for human disease[J]. Genome Res, 9(2): 99-100.
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