[1]赵为民,任守文,方晓敏,等.肌肉中特异表达真核载体的构建及表达特异性验证[J].江苏农业学报,2016,(06):1359-1366.[doi:doi:10.3969/j.issn.1000-4440.2016.06.025]
　ZHAO Wei-min,REN Shou-wen,FANG Xiao-min,et al.Construction and verification of muscle-specific expression eukaryotic vector[J].,2016,(06):1359-1366.[doi:doi:10.3969/j.issn.1000-4440.2016.06.025]
点击复制

肌肉中特异表达真核载体的构建及表达特异性验证()
分享到：

江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2016年06期
页码:
1359-1366
栏目:
畜牧兽医·水产养殖
出版日期:
2017-02-07

文章信息/Info

Title:
Construction and verification of muscle-specific expression eukaryotic vector
作者:
赵为民; 任守文; 方晓敏; 李碧侠; 涂枫; 付言峰; 赵芳; 王学敏
（江苏省农业科学院畜牧研究所/动物品种改良和繁育重点实验室，江苏南京210014）
Author(s):
ZHAO Wei-min; REN Shou-wen; FANG Xiao-min; LI Bi-xia; TU Feng; FU Yan-feng; ZHAO Fang; WANG Xue-min
(Institute of Animal Science, Jiangsu Academy of Agricultural Science/Key Laboratory of Animal Breeding and Reproduction, Nanjing 210014, China)
关键词:
肌肉; 表达载体; MLC; 启动子; DsRed2; 肌细胞
Keywords:
muscle; expression vector; myosin light chain（MLC）; promoter; red fluorescent protein(DsRed2); myoblasts
分类号:
Q782
DOI:
doi:10.3969/j.issn.1000-4440.2016.06.025
文献标志码:
A
摘要:
为构建肌肉中特异表达真核载体，本试验以PGL4.10质粒为骨架，利用基因合成、酶切连接方法获得重组载体，同时利用红色荧光蛋白基因DsRed2作为标记基因来检测重组载体在非肌源细胞和肌细胞中的表达情况。结果表明，成功构建了含有肌球蛋白轻链基因MLC启动子及其增强子的肌肉中特异表达载体，通过mRNA和蛋白水平检测，该表达载体在非肌源性细胞和肌细胞中均不表达DsRed2，只在分化的肌细胞中表达DsRed2。该表达载体的构建为制备肌肉中特异表达的转基因动物奠定了基础。
Abstract:
To construct a muscle-specific eukaryotic expression vector and to verify its specific expression, a recombinant vector was constructed using gene synthesis and enzyme digestion ligation method based on PGL4.10 plasmid as a backbone. Meanwhile, a red fluorescent protein (DsRed2) was used as a marker to detect the expression of the recombinant vector in non-muscle-derived cells and muscle cells. The results showed that the muscle-specific eukaryotic expression vector containing the promoter and enhancer of myosin light chain (MLC) gene was successfully constructed. This expression vector did not express DsRed2 in non-myogenic cells and muscle cells, and could only express in differentiated muscle cells at mRNA and protein levels. The results provide a solid foundation for the preparation of muscle-specific expression for transgenic animal.

参考文献/References:

[1]GORDON J W, SCANGOS G A, PLOTKIN D J, et al. Genetic transformation of mouse embryos by microinjection of purified DNA [J]. Proceedings of the National Academy of Sciences, 1980, 77(12): 7380-7384. 
[2]PALMITER R D, BRINSTER R L, HAMMER R E, et al. Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes [J]. Nature, 1982, 300(5893):611-615.
[3]MAYO K E, HAMMER R E, SWANSON L W, et al. Dramatic pituitary hyperplasia in transgenic mice expressing a human growth hormone-releasing factor gene[J]. Molecular Endocrinology, 1988, 2(7): 606-612.
[4]JI S, HARDY R W, WOOD P A. Transgenic expression of n-3 fatty acid desaturase (fat-1) in C57/BL6 mice: Effects on glucose homeostasis and body weight [J]. Journal of Cellular Biochemistry, 2009, 107(4): 809-817.
[5]LEE S J, MCPHERRON A C. Regulation of myostatin activity and muscle growth [J]. Proceedings of the National Academy of Sciences, 2001, 98(16): 9306-9311.
[6]LAI L, KANG J X, LI R, et al. Generation of cloned transgenic pigs rich in omega-3 fatty acids[J]. Nature Biotechnology, 2006, 24(4): 435-436.
[7]LI T, XU D, ZUO B, et al. Ectopic overexpression of porcine DGAT1 increases intramuscular fat content in mouse skeletal muscle[J]. Transgenic Research, 2013, 22(1): 187-194.
[8]ROSENTHAL N, KORNHAUSER J M, Donoghue M, et al. Myosin light chain enhancer activates muscle-specific, developmentally regulated gene expression in transgenic mice [J]. Proceedings of the National Academy of Sciences, 1989, 86(20): 7780-7784.
[9]GAUTHIER G F, LOWEY S. Distribution of myosin isoenzymes among skeletal muscle fiber types[J]. The Journal of Cell Biology, 1979, 81(1): 10-25.
[10]ROSENTHAL N, BERGLUND E B, WENTWORTH B M, et al. A highly conserved enhancer downstream of the human MLC1/3 locus is a target for multiple myogenic determination factors[J]. Nucleic Acids Research, 1990, 18(21): 6239-6246.
[11]WENTWORTH B M, DONOGHUE M, ENGERT J C, et al. Paired MyoD-binding sites regulate myosin light chain gene expression[J]. Proceedings of the National Academy of Sciences, 1991, 88(4): 1242-1246.
[12]ZHUANG Y, BUTLER B, HAWKINS E, et al. New synthetic Renilla gene and assay system increase expression, reliability and sensitivity[J]. Promega Notes, 2001, 79: 6-11.
[13]BANERJI J, RUSCONI S, SCHAFFNER W. Expression of a β-globin gene is enhanced by remote SV40 DNA sequences[J]. Cell, 1981, 27(2): 299-308.
[14]DONOGHUE M, ERNST H, WENTWORTH B, et al. A muscle-specific enhancer is located at the 3′end of the myosin light-chain 1/3 gene locus[J]. Genes & Development, 1988, 2(12b): 1779-1790.
[15]NISHIZAWA K, KITA Y, KITAYAMA M, et al. A red fluorescent protein, DsRed2, as a visual reporter for transient expression and stable transformation in soybean[J]. Plant Cell Reports, 2006, 25(12): 1355-1361.
[16]SATO Y, IGARASHI Y, HAKAMATA Y, et al. Establishment of Alb-DsRed2 transgenic rat for liver regeneration research[J]. Biochemical and Biophysical Research Communications, 2003, 311(2): 478-481.
[17]SHCHERBO D, MERZLYAK E M, CHEPURNYKH T V, et al. Bright far-red fluorescent protein for whole-body imaging[J]. Nature Methods, 2007, 4(9): 741-746.
[18]WEBER J, WEBEROVA J, CAROBENE M, et al. Use of a novel assay based on intact recombinant viruses expressing green (EGFP) or red (DsRed2) fluorescent proteins to examine the contribution of pol and env genes to overall HIV-1 replicative fitness[J]. Journal of Virological Methods, 2006, 136(1): 102-117.
[19]IRION S, LUCHE H, GADUE P, et al. Identification and targeting of the ROSA26 locus in human embryonic stem cells[J]. Nature Biotechnology, 2007, 25(12): 1477-1482.
[20]MURRAY J D, NANCARROW C D, MARSHALL J T, et al. Production of transgenic merino sheep by microinjection of ovine metallothionein-ovine growth hormone fusion genes[J]. Reproduction, Fertility and Development, 1989, 1(2): 147-155.
[21]RUAN J, LI H, XU K, et al. Highly efficient CRISPR/Cas9-mediated transgene knockin at the H11 locus in pigs [J]. Scientific Reports, 2015, 5:14253.
[22]PLATT R J, CHEN S, ZHOU Y, et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling[J]. Cell, 2014, 159(2): 440-455.

备注/Memo

备注/Memo:
收稿日期：2016-09-22 基金项目：转基因生物新品种培育重大专项（2014ZX08006-003）；国家生猪现代产业技术体系（CARS-36） 作者简介：赵为民（1983-），男，湖北钟祥人，博士，助理研究员，主要从事转基因与育种研究。（Tel）025-84391941；（E-mail）zhao_weimin1983@aliyun.com 通讯作者：王学敏，（Tel）025-84391941；（E-mail）wxm116@sina.com
更新日期/Last Update: 2017-02-07