山羊myostatin基因慢病毒RNA干扰载体的构建及效果验证

Abstract

Abstract: Myostatin gene was an important regulator of muscle growth and development, and the gene mutation or inactivation of the species also displayed the double muscle phenotype of the skeletal hypermuscular, suggesting that modulating myostatin gene expression may allow for increased meat production of livestock. To explore the biological function of myostatin in goat fetal fibroblast and generate the inactivation tool of myostatin, we had cloned the potent targeted site of myostatin into the transfer vector of the lentiviral RNA interference system, constructed the lentiviral mediated RNA interference vector to knockdown myostatin and detected the knockdown efficiency. The results demonstrated that the sequences of RNA interference had been inserted into the transfer vector rightly, and we had constructed the RNA interference of myostatin gene mediated by the lentiviral vector successfully. In addition, we obtained the lentiviral particles by co-transfected of the three plasmids into 293T cells could be transfected into goat fetal fibroblasts with high effective. The results of Real-time PCR demonstrated that the lentiviral vector Lv322 had reduced the myostatin mRNA by 75% (P<0.01), and Western blotting also demonstrated that the protein level of myostatin had been decreased by 94% (P<0.01). The results would provide the important foundation and effective tool to detect the function of myostatin and to generate the inactivation of transgenic animal in the future.

Key words: myostatin gene; goat fetal fibroblasts; lentiviral vector; RNA interference

CLC Number:

LU Jian, ZHANG Shi-fang, ZHANG Xiao-ning, LIU Jia-sen, LI Bi-chun, ZHAO Fu-ping, ZHANG Li, WEI Cai-hong, DU Li-xin. Construction and Detection of the RNA Interference Vector Mediated by Lentiviral to Knockdown Goat myostatin Gene[J]. .

References

1 Clop A, Marcq F, Takeda H, et al. A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep[J]. Nat Genet, 2006, 38: 813~818.

2 Elbashir S M, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[J]. Nature, 2001, 411: 494~498.

3 Furalyov V A, Kravchenko I V, Khotchenkov V P, et al. siRNAs targeting mouse myostatin[J]. Biochemistry (Mosc),2008, 73: 342~345.

4 Glaser S, Anastassiadis K, Stewart A F. Current issues in mouse genome engineering[J]. Nat Genet, 2005, 37: 1187~1193.

5 Hammond S M, Caudy A A, Hannon G J. Post-transcriptional gene silencing by double-stranded RNA[J]. Nat Rev Genet, 2001, 2: 110~119.

6 Jain H, Singh S, Kadam M, et al. Knockdown of the myostatin gene by RNA interference in caprine fibroblast cells[J]. J Biotechnol, 2009, 145: 99~102.

7 Lee S J. Regulation of muscle mass by myostatin[J]. Annu Rev Cell Dev Biol, 2004, 20: 61~86.

8 Lee S J. Sprinting without myostatin: a genetic determinant of athletic prowess[J]. Trends Genet, 2007, 23: 475~477.

9 Li Z B, Kollias H D, Wagner K R. myostatin directly regulates skeletal muscle fibrosis[J].J Biol Chem, 2008, 283: 19371~19378.

10 Lu J, Sun D, Xu L, et al. Selection of an effective small interference RNA to silence myostatin gene expression in sheep fibroblast cells[J]. Biochem Genet, 2012, 51: 1~10.

11 Magee T R, Artaza J N, Ferrini M G, et al. Myostatin short interfering hairpin RNA gene transfer increases skeletal muscle mass[J]. J Gene Med, 2006, 8: 1171~1181.

12 McPherron A C, Lee S J. Double muscling in cattle due to mutations in the myostatin gene[J]. Proc Natl Acad Sci USA, 1997a, 94: 12457~12461.

13 McPherron A C, Lawler A M, Lee S J. Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member[J]. Nature, 1997b, 387: 83~90.

14 Rosen E D, MacDougald O A. Adipocyte differentiation from the inside out[J]. Nat Rev Mol Cell Biol, 2006, 7: 885~896.

15 Rubinson D A, Dillon C P, Kwiatkowski A V, et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference[J]. Nat Genet, 2003, 33: 401~406.

16 Tang D, Zhu H, Wu J, et al. Silencing myostatin gene by RNAi in sheep embryos[J]. J Biotechnol, 2012, 158: 69~74.

17 Tessanne K, Golding M C, Long C R, et al. Production of transgenic calves expressing an shRNA targeting myostatin[J]. Mol Reprod Dev, 2011, 79: 176~185.

18 Tiscornia G, Singer O, Ikawa M, et al. A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA[J]. Proc Natl Acad Sci USA, 2003, 100: 1844~1848.

19 Tripathi A K, Aparnathi M K, Vyavahare, et al. Myostatin gene silencing by RNA interference in chicken embryo fibroblast cells[J]. J Biotechnol, 2012, 160: 140~145.

20 Welle S, Bhatt K, Pinkert C A, et al. Muscle growth after postdevelopmental myostatin gene knockout[J]. Am J Physiol Endocrinol Metab, 2007, 292: E985~E991.

[1]	CHEN Yujing, ZHANG Yiyi, HUANG Zhengyang, SHI Jianzhou, LIU Yangkun, QIU Reng, YAO Lunguang, LI Na. Establishment of BHK-21 Cell Strain for Stably Expressing T7 RNA Polymerase [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(4): 1253-1261.
[2]	ZHANG Yanmin, ZHOU Yanan, CAO Lei, TIAN Yuan, LIU Xuping, TAN Wensong, ZHAO Liang. Expression and Immunogenicity Analysis of Classical Swine Fever Virus E2-GM-CSF Fusion Protein in HEK293T Cells [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(2): 669-676.
[3]	LI Jie, CHEN Jing, YANG Fan, LI Jun, CAI Yafei. Construction and Identification of Lentiviral Overexpression Plasmid of Mouse Zbtb38 Gene [J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(10): 3088-3094.
[4]	QIN Jian-ru, LI Na-ming, XU Chang-jian, LIAO Ming, CAO Wei-sheng. Expression of Enhanced Green Fluorescent Protein (EGFP) Gene Carried by a Lentiviral Vector RCASBP in DF-1 Cell [J]. , 2015, 42(10): 2538-2543.
[5]	HUANG Juan, SHAN Hu. Development and Application of Semi-quantitive RT-PCR for the Main Structural Protein Genes of Porcine Reproductive and Respiratory Syndrome Virus [J]. , 2012, 39(6): 22-26.
[6]	SUN Ke-ning;ZHU Hua-bin;LIN Feng;WANG Dong;HAO Hai-sheng;DU Wei-hua;ZHAO Xue-ming. Development on Lentiviral Vector Construction and Transgentic Animals by Lentiviral Vector [J]. , 2010, 37(8): 116-120.
[7]	ZHANG Rui;GONG Dao-qing. The Progress and Application of Research in Lentiviral Vector [J]. , 2010, 37(6): 227-231.

Construction and Detection of the RNA Interference Vector Mediated by Lentiviral to Knockdown Goat myostatin Gene

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 7

Recommended Articles

Metrics

Comments