利用TALEN技术建立恒河猴TRIM5α基因突变细胞株

生物技术通报 ›› 2016, Vol. 32 ›› Issue (12): 53-57.

利用TALEN技术建立恒河猴TRIM5α基因突变细胞株

王小莉¹，²,余庆²,袁雅红¹，²,腾智平³,李东升²,曾毅¹

1. 北京工业大学生命科学与生物工程学院，北京 100124；
2. 湖北医药学院附属太和医院生命科学研究所，十堰 442000；
3. 中国预防医学科学院病毒所，北京 100052

收稿日期:2016-04-21 出版日期:2016-12-25 发布日期:2016-12-07
作者简介:王小莉，女，博士研究生，研究方向：肿瘤病毒学；E-mail：xiaolitina@126.com
基金资助:
国家科技部重大专项（2013ZX10001004-002-005）

Establishment of Mutagenesis Cell Line of Macaca mulatta Gene TRIM5α by TALEN

WANG Xiao-li¹，²，YU Qing²,YUAN Ya-hong¹，²,TENG Zhi-ping³,LI Dong-sheng²,ZENG Yi¹

1. College of Life Sciences，Beijing University of Technology，Beijing 100124；
2. Life Science Institute，Affiliated Taihe Hospital，Hubei University of Medicine，Shiyan 442000；
3. Institute of Virology，Chinese Academy of Preventive Medicine，Beijing 100052

Received:2016-04-21 Published:2016-12-25 Online:2016-12-07

摘要/Abstract

摘要： 利用TALEN技术建立恒河猴TRIM5α基因突变细胞株，为进一步研究TRIM5α基因的功能奠定基础。构建针对TRIM5α打靶基因的TALEN，通过点突变的方法获得包含TRIM5α第7个外显子中打靶位点1 211-1 224的基因序列并构建点突变donor载体。将打靶TRIM5α基因的TALEN质粒和donor质粒电转入恒河猴肾细胞（LLC-MK2）中，无限稀释法获得单克隆细胞系，通过抽提基因组DNA，再利用PCR技术扩增目标序列并测序筛选出TRIM5α碱基缺失（1 215-1 216）和点突变（1 213-1 215，1 217）的细胞系。通过共转TALEN质粒和点突变的donor质粒筛选获得了TRIM5α基因敲除和TRIM5α基因点突变的LLC-MK2细胞系。

关键词: TRIM5α, TALEN, 恒河猴, 点突变

Abstract: Using transcription activator-like effector nuclease（TALEN）to establish the mutagenesis cell line of Macaca mulatta gene TRIM5α is to lay the foundation for further studying the function of TRIM5α gene. The TALEN plasmids targeting TRIM5α was constructed. The site-directed gene mutagenesis donor vector was obtained by site-directed gene mutagenesis technology and the sequence contained the seventh exon of TRIM5α at the position from 1 211 to 1 224. The TALEN plasmids and donor vector were co-transfected into the M. mulatta’s kidney cell line（LLC-MK2）by electroporation. The single cell line was obtained by the infinite dilution method. By extracting the genomic DNA and amplifying the target sequence by PCR，the cell line with base deletion（1 215-1 216）and site mutations（1 213-1 215，1 217）of TRIM5α was screened. Conclusively，the LLC-MK2 single cell line with knockout and site-directed gene mutagenesis of gene TRIM5α was obtained.

Key words: tripartite motif 5 alpha, TALEN, Macaca mulatta, site-directed gene mutagenesis

中图分类号:

10.13560/j.cnki.biotech.bull.1985.2016.12.010

王小莉,余庆,袁雅红，腾智平,李东升,曾毅. 利用TALEN技术建立恒河猴TRIM5α基因突变细胞株[J]. 生物技术通报, 2016, 32(12): 53-57.

WANG Xiao-li,YU Qing,YUAN Ya-hong,TENG Zhi-ping,LI Dong-sheng,ZENG Yi. Establishment of Mutagenesis Cell Line of Macaca mulatta Gene TRIM5α by TALEN[J]. Biotechnology Bulletin, 2016, 32(12): 53-57.

参考文献

[1] Richman DD, Margolis DM, Delaney M, et al. The challenge of finding a cure for HIV infection[J] . Science, 2009, 323（5919）：1304-1307.
[2] Excler JL, Robb ML, Kim JH. Prospects for a gGlobally effective HIV-1 vaccine[J] . Am J Prev Med, 2015, 49（6 Suppl 4）：S307-318.
[3] Smaglik P. AIDS vaccine researchers turn from chimps to monkeys[J] . The Scientist, 1999, 13（16）：7-9.
[4] Antony JM, MacDonald KS. A critical analysis of the cynomolgus macaque, Macaca fascicularis, as a model to test HIV-1/SIVvaccine efficacy[J] . Vaccine, 2015, 33（27）：3073-3083.
[5] Friedrich BM, Dziuba N, Li G, et al. Host factors mediating HIV-1 replication[J] . Virus Res, 2011, 161（2）：101-114.
[6] Malim MH, Bieniasz PD. HIV restriction factors and mechanisms of evasion[J] . Cold Spring Harb Perspect Med, 2012, 2（5）：a006940.
[7] Sauter D. Counteraction of the multifunctional restriction factor tetherin[J] . Front Microbiol, 2014, 5：163.
[8] Newman RM, Hall L, Connole M, et al. Balancing selection and the evolution of functional polymorphism in Old World monkey TRIM5alpha[J] . Proc Natl Acad Sci USA, 2006, 103（50）：19134-19139.
[9] Puvvada MP, Patel SS. Role of trim5α in the suppression of cross-species transmission and its defence against human immunodefici-ency virus[J] . Curr HIV Res, 2013, 11（8）：601-609.
[10] Sakuma R, Mael AA, Ikeda Y. Alpha interferon enhances TRIM5-alpha-mediated antiviral activities in human and rhesus monkey cells[J] . J Virol, 2007, 81（18）：10201-10206.
[11] Sastri J, Johnsen L, Smolin N, et al. Restriction of HIV-1 by rhe-sus TRIM5α is governed by alpha helices in the Linker2 region[J] . J Virol, 2014, 88（16）：8911-8923.
[12] Li X, Kim J, Song B, et al. Virus-specific effects of TRIM5α（rh）RING domain functions on restriction of retroviruses[J] . J Virol, 2013, 87（13）：7234-7245.
[13] Lukic Z, Goff SP, Campbell EM, et al. Role of SUMO-1 and SUMO interaction motifs in rhesus TRIM5α-mediated restriction[J] . Retrovirology, 2013, 10：10.
[14] Brandariz-Nu?ez A, Roa A, Valle-Casuso JC, et al. Contribution of SUMO- interacting motifs and SUMOylation to the antiretroviral properties of TRIM5α[J] . Virology, 2013, 435（2）：463-471.
[15] Mussolino C, Alzubi J, Fine EJ, et al. TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity[J] . Nucleic Acids Res, 2014, 42（10）：6762-6773.
[16] Lascano J, Uchil PD, Mothes W, et al. TRIM5 retroviral restriction activity correlates with the ability to induce innate immune signaling[J] . J Virol, 2015, 90（1）：308-316.
[17] Kimura T, Jain A, Choi SW, et al. TRIM-mediated precision autophagy targets cytoplasmic regulators of innate immunity[J] . J Cell Biol, 2015, 210（6）：973-989.