Sequencing and Bioinformatic Investigation of Introducing a Repressive Micro-RNA Target Sites in the 3'UTR of Myostatin Gene in some Indigenous Sheep Breeds of Iran

Document Type : Genetics & breeding


1 Department Animal Science, Faculty of agriculture, Ferdowsi University of Mashhad, Mashhad, Iran.

2 Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran.


Introduction[1] Myostatin, known as the growth and differentiation factor 8 (GDF8), is a negative regulator of skeletal muscle growth in mammals. This gene is conserved across mammalian species and expressed in developing as well as mature skeletal muscles fiber cells. It has been demonstrated that a natural mutation in myostatin (MSTN) gene is effective in muscle tissue overgrowth in species such as cattle, sheep, pig and mouse. Later on, with administration of genome editing techniques on rabbit (17), pig (16) and dog (31), double muscling trait was successfully achieved by modifying MSTN gene. It can be inferred from the studies that the removal of MSTN’s inhibitory role will leads to muscle increase, an observation similar to that of other mammalian species. Therefore, it can be considered as a candidate gene for growth and carcass traits. This simple mutation can be used as a model for genetic engineering of farm animals to improve growth traits.
Materials and Methods In total, blood samples of 15 sheep from each breed of Dalagh, Baluchi and Zel were collected. Genomic DNA was extracted from whole blood using Guanidium Thiocyanate-Silica Gel method (Diatom DNA Prep. 100, Isogene, Russia) following the manufacturer’s instruction. The integrity of the extracted DNA was assessed by electrophoresis on a 0.8% agarose gel and the purity of the obtained DNA was evaluated by Epoch microplate spectrophotometer (BioTek, USA). A 2180 bp region from 3UTR of ovine MSTN gene was amplified by standard PCR reaction in a total volume of 25 µl. Tree sets of specific overlapping primers were used to amplify part of the 3UTR region in MSTN gene (Table 1). The PCR products then purified by ethanol precipitation method (14) and sequenced. The sequencing results homology were checked by BLAST and assembled using CLC sequence viewer 8.0. Then miRNA target sites were analyzed to create a potent in silico modification which serve as a target site for the microRNAs miR-1 and miR-206 with suppressing effect on MSTN transcript. At the end to assess the formation of any new and undesired motif due to the creation of our in silico modification, the whole area analyzed with the motif finder application.
Results and Discussion This study was performed to identify and compare DNA sequence of a 2180 bp region from the 3'UTR of myostatin gene in Dalagh, Baluchi and Zel sheep breeds and with the aim of introducing an in silico modification to introduce a mutation with a positive impact on growth rate. Results showed that there was a high similarity between 3' UTR sequences of GDF8 gene in Zel, Dalagh and Baluchi sheep breeds. All samples were monomorphic and had the g+6723G allele, which do not cause double muscle phenotype (Figure 2). An in silico approach employed to modify the 3'UTR of the myostatin gene in this indigenous sheep breeds in order to create miR-1 and miR-206 (ACATTCCA) target sequences naturally occurring in the Texel sheep (Figure 3). After applying these changes, the possibility of creating unwanted new regulatory elements was investigated using the motif finder software. The results showed that the introduced mutations did not create any new motifs that had a known regulatory role in mammals.  It was demonstrated that this mutation can attribute to 38% of the additive genetic variance for muscle depth in the Charollais lambs (12). In another study, this mutation found to have a significant increase in muscle mass and reduced carcass fat in Norwegian White sheep (6). Therefore, this single modification can be considered as the best mutation for double muscling due to its large effect on the muscling phenotype.
Conclusion Due to the large effect that g+6723G>A mutation has on the phenotype double muscle and also the absence of other known effects on the phenotypes, this mutation could be considered as one of the best candidates for genome editing that can create indigenous sheep with overgrown muscle phenotype in the future by using of genetic engineering techniques. It is feasible to introduce this mutated allele by genetic engineering methods as a desirable genetic modification for improving indigenous sheep breeds. Advantages of using this approach include increasing the genetic progress of breeding programs in compare to traditional methods and maintaining the environmental compatibility of indigenous sheep breeds.


1- Abbasi, V., A. Javadmanesh, and M. Nassiry. 2016. Prediction and in silico validation of expressed micro- RNAs in ovine chromosomes 20. Proceedings of The 2nd International and The 14th Iranian Genetics Congress. 21-23 May, Tehran, Iran. (In Persian).
2- Aslaminejad, A. A. Nassiry, M. R. Shahroudi, F. E. Valizadeh, R. Javadmanesh, A. Norouzy, A. Samei, A. and Ghiasi, H. 2006. Study on the genetic polymorphisms of candidate genes in Karakul. Agricultural Sciences and Technology 20 (4): Pe21-Pe29. (In Persian).
3- Bartel, D. P. 2004. MicroRNAs: genomics, biogenesis, mechanism, and function: a review. Cell, 116: 281–297.
4- Bartel, D. P. 2009. MicroRNAs: target recognition and regulatory Functions. A review. Cell, 136: 215–233.
5- Bellinge, R. H., D. A. Liberles, S. P. Iaschi, and P. A. O’Brien. 2005. Myostatin and its implications on animal breeding: a review. Animal Genetics, 36: 1-6.
6- Boman, I. A., G. Klemetsdal, O. Nafstad, T. Blichfeldt, and D. I. Våge. 2010. Impact of two myostatin (MSTN) mutations on weight gain and lamb carcass classification in Norwegian White Sheep (Ovis aries). Genetics Selection Evolution, 42: 4.
7- Chen, J. F; M. M. Elizabeth, J. M. Thomson, Q. Wu, T. E. Callis, S. M. Hammond, F. L. Conlon, D. Z. Wang. 2005. The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nature Genetics, 38: 228-233.
8- Chris P., D. F. Carlson, R. Huddart, C. R. Long, J. H. Pryor, T. J. King, S. G. Lillico, A. J. Mileham, D. G. McLaren, C. B. A. Whitelaw, and S. C. Fahrenkrug. 2015. Genome edited sheep and cattle. Transgenic Research, 24: 147-153.
9- Clop, A., F. Marcq, H. Takeda, D. Pirottin, X. Tordoir, B. Bibe, J. Bouix, F. Caiment, J. M. Elsen, F. Eychenne, C. Larzul, E. Laville, F. Meish, D. Milenkovic, J. Tobin, and C. M. Charlier. 2006. A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep. Nature Genetics, 38: 813-818.
10- Gonzalez-C. N., W. E. Taylor, K. E. Yarasheski, I. Sinha- Hikim, K. Ma, S. Ezzat, R. Shen, R. Lalani, S. Asa, M. Mamita, G. Nair, S. Arver, and S. Bhasin. 1998. Organization of the human myostatin gene and expression in healthy men and HIV-infected men with muscle wasting. Proceedings of the National Academy of Sciences of the United States of America, 95: 14938–14943.
11- Grisolia, A. B., G.T. D’Angelo, L. R. Porto Neto, and F. Siqueira. 2009. Myostatin (GDF8) single nucleotide polymorphisms in Nellore cattle. Genetics and Molecular Research, 8: 822-830.
12- Hadjipavlou, G., O. Matika, A. Clop, and S. C. Bishop. 2008. Two single nucleotide polymorphisms in the myostatin (GDF8) gene have significant association with muscle depth of commercial Charollais sheep. Animal Genetics, 39: 346–353.
13- Heravi Mousavi, A., M. Ahouei, M.R. Nassiry and A. Javadmanesh. 2006. Association of leptin polymorphism with production, reproduction and plasma glucose level in Iranian Holstein cows. Asian Australian Journal of Animal Sciences. 19 (5): 627-631.
14- Javadmanesh, A. 2013. Contribution of IGF system and GH-IGF1 axis to heterosis in a bovine fetus model. PhD thesis. The University of Adelaide, Adelaide, Australia.
15- Javadmanesh, A., M. R. Nassiry, and M. Azghandi. 2017. Sequencing of HVR-III region of mtDNA in Iranian sheep breeds. Journal of Animal Science Researches, 27 (2): 133-141. (In Persian).
16- Lillico, S. G., C. Proudfoot, D. F. Carlson, D. Stverakova, C. Neil, C. Blain, T. J. King, W. A. Ritchie, W. Tan, A. J. Mileham, D. G. McLaren, S. C. Fahrenkrug, and C. B. A. Whitelaw. 2013. Live pigs produced from genome edited zygotes. Scientific Reports, 3: 2847.
17- Lv, Q., L. Yuan, J. Deng, M. Chen, Y. Wang, J. Zeng, Z. Li, and L.Lai, 2016. Efficient generation of myostatin gene mutated rabbit by CRISPR/Cas9. Scientific Reports, 6: 25029.
18- McPherron, A, and S. J. Lee. 1997. Double muscling in cattle due to mutations in the myostatin gene. Proceedings of the National Academy of Sciences of the United States of America, 94: 12457–12461.
19- McPherron, A. C., A. M. Lawler, and S. J. Lee. 1997. Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature, 387: 83–90.
20- Miar, Y., R. Salehi, S. A. Aleyasi, D. Kolbehdari, and S. Raoofzadeh. 2009. Polymorphisms in Myostatin Gene and its Association with Growth and Carcass Traits in Iranian Sheep. The 6th National Biotechnology Congress of Iran. (In Persian).
21- Nassiry, M. R., M. Tahmoorespur, A. Javadmanesh, M. Soltani, and S. Foroutanifar. 2006. Calpastatin polymorphism and its association with daily gain in Kurdi sheep. Iranian Journal of Biotechnology 4 (3): 188-192.
22- Nassiry, M. R., F. Eftekhari Shahroudi, M. Tahmoorespur and A. Javadmanesh. 2008. The diversity of BoLA-DRB3 gene in Iranian native cattle. Asian Australian Journal of Animal Sciences. 21 (4): 456-470.
23- Ran, F. A., P. D. Hsu, J. Wright, V. Agarwala, D. A Scott, and F. Zhang. 2013. Genome engineering using the CRISPR-Cas9 system. Nature Protocols, 8: 2281-2308.
24- Rhoades, M. W., B. J. Reinhart, L.P. Lim, C. B. Burge, B. Bartel, and D. P. Bartel. 2002. Prediction of Plant MicroRNA Targets. Cell, 110: 513–520.
25- Tang, D. Z., H. J. Wu, H. Chen, Y. Zhang, X. Zhao, X. Chen, W. Du, D. Wang, and X. Lin. 2012. Silencing myostatin gene by RNAi in sheep embryos. Journal of Biotechnology, 158: 69–74.
26- Taylor, W. E., S. Bhasin, J. A. Frances Byhower, M. Azam, H. Darril, J. r. Willard, F. C. Kull, and N. Gonzalez-Cadav. 2001. Myostatin inhibits cell proliferation and protein synthesis in C2C12 muscle cells. American Journal of Physiology, Endocrinology and Metabolism, 280: E221–E228.
27- Warner, R. D., P. L. Greenwood, and D. M. Ferguson. 2011. Understanding genetic and environmental effects for assurance of meat quality. Control of Meat Quality, 117-145.
28- Yousefi, A. R., Kohram, H. Zare Shahneh, A. Nik-khah, A. and Campbell, A. W. 2012. Comparison of the meat quality and fatty acid composition of traditional fat-tailed (Chall) and tailed (Zel) Iranian sheep breeds. Meat Science. 92 (4): 417-422.
29- Zhang, Z. J., Y.H. Ling, L. J. Wang, Y. F. Hang, X. F. Guo, Y. H. Zhang, J. P. Ding, and X. R. Zhang. 2013. Polymorphisms of the myostatin gene (MSTN) and its relationship with growth traits in goat breeds. Genetics and Molecular Research, 12: 965-971.
30- Zheng, Y., H. Ma, Y. Zheng, Y. Wang, B. Zhang, X. He, X. He, J. Liu, and Y. Zhang. 2012. Site-directed mutagenesis of the myostatin gene in ovine fetal myoblast cells in vitro. Research in Veterinary Science. 93 (2): 763-769.
31- Zou, Q., X. Wang, Y. Liu, Z. Ouyang, H. Long, S. Wei, J. Xin, B. Zhao, S. Lai, J. Shen, Q. Ni, H. Yang, H. Zhong, L. Li, M. Hu, Q. Zhang, Z. Zhou, J. He, Q. Yan, N. Fan, Y. Zhao, Z. Liu, L. Guo, J. Huang, G. Zhang, J. Ying, L. Lai, X. Gao. 2015. Generation of gene-target dogs using CRISPR/Cas9 system. Journal of Molecular Cell Biology. 7 (6): 580-583.