Transfection of bovine spermatogonial stem cells in vitro

Document Type: Full paper (Original article)

Authors

1 Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

2 Department of Biotechnology, Iranian Research Organization for Science and Technology, Tehran, Iran

3 Post-Graduates, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

4 Department of Clinical Science, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

Abstract

Spermatogonial stem cells (SSCs) are the only stem cells in adults that can transfer genetic information to the future generations. Considering the fact that a single SSC gives rise to a vast number of spermatozoa, genetic manipulation of these cells is a potential novel technology with feasible application to various animal species. The aim of this study was to evaluate enhanced green fluorescent protein (EGFP) gene transfection into bovine SSCs via liposome carrier and assess the best incubation day in uptake exogenous gene by SSCs. Transfection efficiency of EGFP gene with lipofectamine 2000 was determined in days following each three day of transfection (day 4, 6 and 8 of the culture) by fluorescent microscope. Results showed that the transfected cells through lipofection increased significantly (P<0.05) in each three days of transfection in comparison with those of the control groups. The transfected SSCs were higher in comparison with those of the free exogenous gene carrier groups (P<0.05). In comparison with these three days, the rate of infected cells was higher when transfection proceeds at day four. It was concluded that lipofectamine can be used safely for direct loading exogenous DNA to SSCs particularly during the fourth day of culture.

Keywords


Anway, MD; Folmer, J and Wright, WW (2003). Isolation of sertoli cells from adult rat testes: an approach to ex vivo studies of sertoli cell function. Biol. Reprod., 68: 996-1002.

Bellue, AR; Caricchia, JC; Millette, CF; O’Brien, DA; Bhatnagar, YM and Dym, M (1977). Spermatogonic cells of the prepubertal mouse. Isolation and characterization. J. Cell. Biol., 74: 1-9.

Brinster, RL and Avarbock, MR (1994). Germline trans-mission of donor haplotype following spermatogonial transplantation. Proc. Natl. Acad. Sci., 91: 11303-11307.

Dirami, G; Ravindranath, N; Pursel, V and Dym, M (1999). Effects of stem cell factor and granulocyte macrophage-colony stimulating factor on survival of porcine type A spermatogonia culture in KSOM. Biol. Reprod., 61: 225-230.

Felgner, PL; Gadek, TR; Holm, M and Roman, R (1987). Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Sci., 84: 7413-7417.

Garrett, FE; Goel, SH; Yasul, J and Koch, RA (1999). Liposome fuse with sperm cells and induce activation by delivery of important agents. BBA. 1417: 77-88.

Izadyar, F; Creemers, LB and Van Dissel-Emiliani, FM (2000). Spermatogonial stem cell transplantation. Mol. Cell Endocrinol., 169: 21-26.

Izadyar, F; Sepierenberg, GT; Creemers, LB; Ouden, K and Rooij, DG (2002). Isolation and purification of type Aspermatogonia from the bovine testis. Reproduction. 124: 85-94.

Kubota, H and Brinster, RL (2008). Culture of rodent spermatogonial stem cells, male germline stem cells of the postnatal animal. Methods Cell Biol., 86: 59-84.

Lai, Y; Drobinskaya, I and Chuguang, L (2008). Genetic modification of cells for transplantation. Adv. Drug Deliv. Rev., 60: 146-159.

Nagona, M; Clayton, J; Kley, E and Mary, R (2001). Transgenic mice produced by retroviral transduction of male germ-line stem cells. PNAS., 98: 13090-13095.

Niu, Y and Liang, SH (2008). Progress in gene transfer by germ cells in mammals. J. Genet. Genomics. 35: 701-714.

Ogachi, S; Kamihira, M; You, J; Tachibana, A and Lijima, Sh (1998). Exogene gene transfection into quail embryo using cationic lipid vesicles. J. Ferment. Bioeng., 86: 118-120.

Sciamanna, I (2002). DNA dose and sequence dependence in sperm-mediated gene transfer. Mol. Reprod. Dev., 56: 301-305.

Whitelaw, CB and Lilico, SG (2008). Production of transgenic farm animals by viral vector-mediated gene transfer. Reprod. Domest. Anim., (Suppl. 43), 2: 355-389.

Xiang-Yang, M (2011). Production of transgenic animals using spermatogonial stem cells. Agri. Sci. in China. 10: 762-768.

Younezawa, T; Furahata, Y; Hirabayashi, K; Suzuki, M and Yamanouchi, K (2002). Protamine-derived synthetic enhanced the efficiency of sperm-mediated gene transfer using liposome-peptid-DNA complex. J. Reprod. Dev., 48: 281-286.