Validation of Gene Silencing Using RNA Interference in Buffalo Granulosa Cells

  • Monga, Rachna (Molecular Endocrinology Laboratory, National Dairy Research Institute) ;
  • Datta, Tirtha Kumar (Animal Biochemistry Division, Animal Biotechnology Center, National Dairy Research Institute) ;
  • Singh, Dheer (Molecular Endocrinology Laboratory, National Dairy Research Institute)
  • Received : 2011.04.07
  • Accepted : 2011.07.19
  • Published : 2011.11.01


Silencing of a specific gene using RNAi (RNA interference) is a valuable tool for functional analysis of a target gene. However, information on RNAi for analysis of gene function in farm animals is relatively nil. In the present study, we have validated the interfering effects of siRNA (small interfering RNA) using both quantitative and qualitative gene silencing in buffalo granulosa cells. Qualitative gene knockdown was validated using a fluorescent vector, enhanced green fluorescence protein (EGFP) and fluorescently labeled siRNA (Cy3) duplex. While quantitatively, siRNA targeted against the luciferase and CYP19 mRNA was used to validate the technique. CYP19 gene, a candidate fertility gene, was selected as a model to demonstrate the technique optimization. However, to sustain the expression of CYP19 gene in culture conditions using serum is difficult because granulosa cells have the tendency to luteinize in presence of serum. Therefore, serum free culture conditions were optimized for transfection and were found to be more suitable for the maintenance of CYP19 gene transcripts in comparison to culture conditions with serum. Decline in fluorescence intensity of green fluorescent protein (EGFP) was observed following co-transfection with plasmid generating siRNA targeted against EGFP gene. Quantitative decrease in luminescence was seen when co-transfected with siRNA against the luciferase gene. A significant suppressive effect on the mRNA levels of CYP19 gene at 100 nM siRNA concentration was observed. Also, measurement of estradiol levels using ELISA (enzyme-linked immunosorbent assay) showed a significant decline in comparison to control. In conclusion, the present study validated gene silencing using RNAi in cultured buffalo granulosa cells which can be used as an effective tool for functional analysis of target genes.


  1. Ackermann, K., J. Fauss and W. Pyerin. 1994. Inhibition of cyclic AMP-triggered aromatase gene expression in human choriocarcinoma cells by antisense oligodeoxynucleotide. Can. Res. 54:4940-4946.
  2. Adashi, E. Y., C. E. Resnick, A. M. Brodie, M. E. Svoboda and J. J. Van Wky. 1985. Somatomedin-C mediated potentiation of follicle stimulating hormone-induced aromatase activity of cultured rat granulosa cells. Endocrinology 117:2313-2320.
  3. Adashi, E. Y., C. E. Resnick, E. R. Hernandez, J. V. May, M. Kecht, M. E. Svoboda and J. J. Van Wyk. 1988. Insulin-like growth factor-I as an amplifier of follicle stimulating hormone action: studies on mechanism(s) and site(s) of action in cultured rat granulosa cells. Endocrinology 122:1583-1591.
  4. Aravin, A. A., M. S. Klenov, V. V. Vagin, Ya. M. Rozovskii and V. A. Gvozdev. 2002. Role of double-stranded RNA in eukaryotic gene silencing. Mol. Biol. 36(2):180-188.
  5. Balasubramanian, K., H. A. Lavoie, J. C. Garmey, D. M. Stocco and J. D. Veldhius. 1997. Regulation of porcine granulosa cell steroidogenic acute regulatory protein (StAR) by IGF1: synergism with FSH or protein kinase A agonist. Endocrinology 138:433-439.
  6. Brandt, M. E., D. Puett and S. J. Zimniski. 1990. Divergence between ovarian aromatase activity, estrogen, and androgen levels in the cycling rat. Endocrinology 126:72-79.
  7. Bruning- Richardson, A. and G. A. McConkey. 2005. RNAi in the malaria parasite plasmodium. In: Gene Silencing by RNA Interference Technology and Application (Ed. M. Sohail).
  8. Conley, A. and M. Hinshelwood. 2001. Mammalian aromatases. Reprod. 121:685-695.
  9. Drost M. 2007. Bubaline versus bovine reproduction. Theriogenology 68:447-449.
  10. EI-Wishy, A. B. 2007. The postpartum buffalo. I. Endocrinological changes and uterine involution. Theriogenology 97:201-215.
  11. Evans, A. C. O., J. L. H. Ireland, M. E. Winn, P. Lonergan, G. W. Smith, P. M. Coussens and J. J. Ireland. 2004. Identification of genes involved in apoptosis and dominant follicle development during follicular waves in cattle. Biol. Reprod. 70:1475-1484.
  12. Fire, A. and M. Nirenberg. 2005. RNA interference technology: From basic science to drug development. Cambridge University Press, Cape Town.
  13. Ghai, S., R. Monga, T. K. Mohanty, M. S. Chuahan and D. Singh. 2010. Tissue-specific promoter methylation coincides with Cyp19 gene expression in buffalo (Bubalus bubalis) placenta of different stages of gestation. Gen. Comp. Endocrinol. 169: 182-189.
  14. Gonzalez, G., L. Pfannes, R. Brazas and R. Straiker. 2007. Selection of an optimal transfection reagent and comparison to electroporation for the delivery of viral RNA. J. Virol. Methods 145(1):14-21.
  15. Guerra-Crespo, M., J. L. Charli, V. H. Ia, G. Pedraza-Alva and Lerez-Martinez. 2003. Polyethylenimine improves the transfection efficiency of primary cultures of post-mitotic rat fetal hypothalamic neurons. J. Neurosci. Methods 127:179-192.
  16. Gutierrez, C. G., B. K. Campbell and R. Webb. 1997. Development of a long-term bovine granulosa cell culture system: Induction and maintenance of estradiol production, response to follicle stimulating hormone, and morphological characteristics. Biol. Reprod. 56:608-616.
  17. Hickey, G. J., R. B. Oonk, P. F. Hall and J. S. Richards. 1989. Aromatase cytochrome P450 and cholesterol side-chain cleavage cytochrome P450 in corpora lutea of pregnant rats: diverse regulation of peptide and steroid hormones. Endocrinology 125:1673-1682.
  18. Hirano, T., N. Yamauchi, F. Sato, T. Soh and M. Hattori. 2004. Evaluation of RNA interference in developing porcine granulosa cells using fluorescence reporter genes. Reprod. Dev. 50:599-603.
  19. Holen, T., M. Amarzguioui, M. T. Wiiger, E. Babaie and H. Prydz. 2002. Positional effects of short interfering RNAs targeting the human coagulation trigger tissue factor. Nucleic Acids Res. 30:1757-1766.
  20. Khatri, K., A. Rawat, S. Mahor, P. N. Gupta and S. P. Vyas. 2006. Double stranded RNAs as gene silencing tool: An overview. Indian J. Biotech. 5:460-470.
  21. Kobayashi, S., M. Sakatani, S. Kobayashi, K. Okuda and M. Takahashi. 2007. Gene silencing of cyclooxygenase-2 mRNA by RNA interference in bovine Cumulus-Granulosa cells. J. Reprod. Dev. 53:1305-1311.
  22. Kumar, R., D. S. Conklin and V. Mittal. 2003. High-throughput selection of effective RNAi probes for gene silencing. Genome Res. 13:2333-2340.
  23. Lambeth, L. S., T. G. Wise, M. S. Moore and T. J. Doran. 2006. Comparison of bovine RNA polymerase III promoters for short hairpin RNA expression. Anim. Genet. 37:369-372.
  24. Lobo, J. O. and F. L. Bellino. 1989. Estrogen synthetase (Aromatase) activity in primary culture of human term placental cells: Effects of cell preparation, growth medium, and serum on adenosine 3', 5'-Monophosphate response. J. Clin. Endocrinol. Metabol. 69:868-874.
  25. Manik, R. S., P. Palta, S. K. Singla and V. Sharma. 2002. Folliculogenesis in buffalo (Bubalus bubalis): a review. Reprod. Fertil. Dev. 14(5):315-325.
  26. May, J. M. and D. W. Schomberg. 1981. Granulosa cell differentiation in vitro: Effect of insulin on growth and functional integrity. Biol. Reprod. 25:421-431.
  27. Monga, R., S. Ghai, T. K. Datta and D. Singh. 2011. Tissuespecific promoter methylation and histone modification regulate CYP19 gene expression during folliculogenesis and luteinization in buffalo ovary, (Article in press).
  28. Nimz, M., M. Spitschak, F. Schneider, R. Furbass and J. Vanselow. 2009. Down- regulation of genes encoding steroidogenic enzymes and hormone receptors in late preovulatory follicles of the cow coincides with an accumulation of intrafollicular steroids. Domest. Anim. Endocrinol. 37(1):45-54.
  29. Onteru, S. K., D. Sharma, D. Singh and M. K. Sharma. 2008. CYP19 (cytochrome P450 aromatase) gene polymorphism in murrah buffalo heifers of different fertility performance. Res. Vet. Sci. 87:427-437.
  30. Shanmugam, M., S. Pandita and P. Palta. 2009. Effects of FSH and LH on steroid production by buffalo (Bubalus bubalis) granulosa cells cultured in vitro under serum free conditions. Reprod. Domest. Anim. 45:922-926.
  31. Sharma, D., S. Ghai and D. Singh. 2009. Different promoter usage for CYP19 gene expression in buffalo ovary and placenta. Gen. Comp. Endocrinol. 162:319-328.
  32. Silva, J. M. and C. A. Price. 2000. Effect of follicle-stimulating hormone on steroid secretion and messenger ribonucleic acids encoding cytochromes P450 aromatase and cholesterol sidechain cleavage in bovine granulosa cells in vitro. Biol. Reprod. 62:186-191.
  33. Sourdaine, P., P. Mullen, R. White, J. Telford, M. G. Parker and W. R. Miller. 1996. Aromatase activity and CYP19 gene expression in breast cancers. J. Steroid Biochem. Mol. Biol. 59:191-198.
  34. Tezano, G. M. 2005. Follicular dynamics and reproductive technologies in buffalo. In: Buffalo production and Research. Rome: REU technical Series 67. pp. 109-136.
  35. Urban, R. J., J. C. Garmey, M. A. Shupnik and J. D. Veldhuis. 1990. Insulin-like growth factor type-I increase concentrations of mRNA encoding CYP450scc enzyme in primary cultures of porcine granulosa cells. Endocrinology 127:2481-2488.
  36. Veldhuis, J. D., R. J. Rodges, A. Dee and E. R. Simpson. 1986. The insulin-like growth factor, somatomedin C, induces the synthesis of cholesterol side-chain cleavage cytochrome P450 and adrenodoxin in ovarian cells. J. Biol. Chem. 261:2499-2502.
  37. Xu, Z. Z., H. A. Garverick, G. W. Smith, M. F. Smith, S. A. Hamilton and R. S. Youngquist. 1995. Expression of messenger ribonucleic acid encoding cytochrome P450 sidechain cleavage, cytochrome P450 17a-hydroxylase,and cytochrome P450 aromatase in bovine follicles during the first follicular wave. Endocrinology 136:981-989.

Cited by

  1. vol.119, pp.10, 2018,