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Quantitative Evaluation of Myostatin Gene in Stably Transfected Caprine Fibroblast Cells by Anti-Myostatin shRNA

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Abstract

Skeletal muscle is the major component of lean tissue that is used for consumption, and myostatin is a negative regulator of skeletal muscle growth. Downregulation of this gene therefore offers a strategy for developing superior animals with enhanced muscle growth. Knockdown of myostatin was achieved by RNA interference technology. The anti-myostatin shRNA were designed and stably transfected in caprine fibroblast cells. The reduced expression of target gene was achieved and measured in clonal fibroblast cells by real-time PCR. Two single-cell clones induced significant decrease of myostatin gene expression by 73.96 and 72.66 %, respectively (P < 0.05). To ensure the appropriate growth of transfected cell, seven media were tested. The best suited media was used for transfected fibroblast cell proliferation. The findings suggest that shRNA provides a novel potential tool for gene knockdown and these stably transfected cells can be used as the donor cells for animal cloning.

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References

  1. Conlon, I., & Raff, M. (1999). Size control in animal development. Cell, 96, 235–244.

    Article  CAS  Google Scholar 

  2. Langley, B., Thomas, M., Bishop, A., Sharma, M., Gilmour, S., & Kambadur, R. (2002). Myostatin inhibits myoblast differentiation by down-regulating myod expression. The Journal of Biological Chemistry, 277, 49831–49840.

    Article  CAS  Google Scholar 

  3. McPherron, A. C., & Lee, S. J. (1997). Double muscling in cattle due to mutations in the myostatin gene. Proceedings of the National Academy of Sciences, 94(23), 12457–12461.

    Article  CAS  Google Scholar 

  4. Grobet, L., Poncelet, D., Royo, L. J., Brouwers, B., Pirottin, D., Michaux, C., Me’nissier, F., Zanotti, M., Dunner, S., & Georges, M. (1998). Molecular definition of an allelic series of mutations disrupting the myostatin function and causing double-muscling in cattle. Mammalian Genome, 9, 210–213.

    Article  CAS  Google Scholar 

  5. Agrawal, N., Dasaradhi, P. V. N., Mohmmed, A., Malhotra, P., Bhatnagar, R. K., & Mukherjee, S. K. (2003). RNA interference: biology, mechanism, and applications. Microbiology and Molecular Biology Reviews, 67(4), 657–685.

    Article  CAS  Google Scholar 

  6. Stewart, C. K., Li, J., & Golovan, S. P. (2008). Adverse effects induced by short hairpin RNA expression in porcine fetal fibroblasts. Biochemical and Biophysical Research Communications, 370(1), 113–117.

    Article  CAS  Google Scholar 

  7. Jain, H., Singh, S., Kadam, M., & Sarkhel, B. C. (2010). Knockdown of the myostatin gene by RNA interference in caprine fibroblast cells. Journal of Biotechnology, 145(2), 99–102.

    Article  CAS  Google Scholar 

  8. Singh, N. K., Singh, S., Jain, S. K., & Sarkhel, B. C. (2012). Evaluation of interferon response induced by anti-myostatin shRNA constructs in goat (Capra hircus) fetal fibroblasts by quantitative real time-polymerase chain reaction. Animal Biotechnology, 23(3), 174–183.

    Article  CAS  Google Scholar 

  9. Boquest, A. C., Grupen, C. G., Harrison, S. J., McIlfatrick, S. M., Ashman, R. J., d’Apice, A. J., & Nottle, M. B. (2002). Production of cloned pigs from cultured fetal fibroblast cells. Biology of Reproduction, 66, 1283–1287.

    Article  CAS  Google Scholar 

  10. Sharma, A., Naziruddin, B., Cui, C., Martin, M. J., Xu, H., Wan, H., Lei, Y., Harrison, C., Yin, J., Okabe, J., Mathews, C., Stark, A., Adams, C. S., Houtz, J., Wiseman, B. S., Byrne, G. W., & Logan, J. S. (2003). Pig cells that lack the gene for alpha1-3 galactosyltransferase express low levels of the gal antigen. Transplantation, 75, 430–436.

    Article  CAS  Google Scholar 

  11. Takeda, K., Tasai, M., Iwamoto, M., Akita, T., Tagami, T., Nirasawa, K., Hanada, H., & Onishi, A. (2006). Transmission of mitochondrial DNA in pigs and progeny derived from nuclear transfer of Meishan pig fibroblast cells. Molecular Reproduction and Development, 73, 306–312.

    Article  CAS  Google Scholar 

  12. Freshney, R. I. (2005). Culture of animal cells: a manual of basic technique. New York:Wiley-Liss.

    Book  Google Scholar 

  13. Daniel, S. M., Raipuria, P., & Sarkhel, B. C. (2008). Efficiency of cloned embryo production using different types of cell donor and electric fusion strengths in goats. Small Ruminant Research, 77, 45–50.

    Article  Google Scholar 

  14. Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real time quantification PCR and the 2 -ΔΔct method. Methods, 25, 402–408.

    Article  CAS  Google Scholar 

  15. Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., & Tuschl, T. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 411, 494–498.

    Article  CAS  Google Scholar 

  16. Kaltenbach, J. P., Kaltenbach, M. H., & Lyons, W. B. (1958). Nigrosin as a dye for differentiating live and dead ascites cell. Experimental Cell Research, 15, 112–117.

    Article  CAS  Google Scholar 

  17. Kung, M., Stadelmann, B., Brodbeck, U., & Butikofer, P. (1997). Addition of G418 and other aminoglycoside antibiotics to mammalian cells results in the release of GPI-anchored proteins. FEBS Letters, 409(3), 333–338.

    Article  CAS  Google Scholar 

  18. Zou, X., Wang, Y., Chang, Y., Yang, Y., et al. (2002). Generation of cloned goat from transfected foetal fibroblast cells, the effect of donor cell cycle. Molecular Reproduction and Development, 61(2), 164–172.

    Article  CAS  Google Scholar 

  19. Melo, E.O., Sousa, R.V., Iguma, L.T., Franco, M.M., Rech, E.L., & Rumpf, R. (2005). Isolation of transfected fibroblast clones for use in nuclear transfer and transgene detection in cattle embryos. Genetics and Molecular Research, 4(4), 812–821.

  20. Yu, J. N., Ma, S. F., Miao, D. Q., Tan, X. W., Liu, X. Y., Lu, J. H., & Tan, J. H. (2006). Effect of cell cycle status on the efficiency of liposome mediated gene transfection in mouse fetal fibroblasts. Journal of Reproduction and Development, 52(3), 373–382.

    Article  CAS  Google Scholar 

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Acknowledgments

The authors are thankful to National Agriculture Innovation Project, Indian council of Agricultural Research, Government of India, New Delhi, for the financial support.

Conflict of Interest

The authors declare that they have no competing interests.

Compliance with Ethical Standards

This is to state that this entire research work does not involve any human or animal.

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Authors and Affiliations

  1. Animal Biotechnology Centre, Nanaji Deshmukh Veterinary Science University, JNKVV Campus, Krishi Nagar, Adhartal, Jabalpur, Madhya Pradesh, 482004, India

    Sudhir Kumar Jain, Hemlata Jain, Dharmendra Kumar & Bikash Chandra Sarkhel

  2. M.P Private Universities Regulatory Commission, Bhopal, M.P, India

    Akhilesh Kumar Pandey

  3. Aquatic Animal Health, Central Institute of Fisheries Education, Mumbai, India

    Megha Kadam Bedekar

Authors
  1. Sudhir Kumar Jain
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  2. Hemlata Jain
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  3. Dharmendra Kumar
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  4. Megha Kadam Bedekar
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  5. Akhilesh Kumar Pandey
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  6. Bikash Chandra Sarkhel
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Correspondence to Sudhir Kumar Jain.

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Jain, S.K., Jain, H., Kumar, D. et al. Quantitative Evaluation of Myostatin Gene in Stably Transfected Caprine Fibroblast Cells by Anti-Myostatin shRNA. Appl Biochem Biotechnol 177, 486–497 (2015). https://doi.org/10.1007/s12010-015-1757-1

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  • DOI: https://doi.org/10.1007/s12010-015-1757-1

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