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Genetic modification, in vitro culture and cryopreservation of bovine spermatogonial stem cells
Spermatogonial stem cells (SSCs) possess the unique capacity of self-renewal and differentiation and thereby can transmit genetic information to the next generation. Combination with techniques such as isolation, culture, preservation, and transplantation of the SSC has facilitated the efficient method for production of transgenic animals, and preservation of livestock and endangered species. The purpose of this study was to genetically modify enriched populations of pre-pubertal germ cells using lentiviral transduction and to develop an efficient in vitro culture system and cryopreservation technique for bovine SSCs. To maximize the efficiency of genetic modification of bovine SSCs, effective enrichment techniques need to be developed. Selection of bovine SSCs using a combination of laminin and gelatin was resulted in a 8-fold enrichment. Selected cells were then transduced using a lentiviral vector containing the transgene for the enhanced green fluorescent protein. Transduction efficiency was 17%. Next, to enhance the efficiency of proliferation for in vitro culture, the effects of various culture conditions and growth factors on bovine cell proliferation were evaluated. Based on the results, we developed the optimal culture conditions [2× rat sertum free medium (rSFM) containing 0.1% FBS together with GDNF, GFRα1, bFGF, EGF, LIF, and CSF-1] for maintaining bovine SSCs over 3 months without any alteration of stem cell characteristics and functions. Also, to develop an effective cryopreservation technique for bovine SSCs, the effects of different freezing methods and various cryoprotective agents were tested. The recovery rate, and proliferation capacity of bovine SSCs were significantly greater for germ cells frozen using tissue freezing methods compared to cell freezing methods. Cryopreservation in the presence of 200 mM trehalose resulted in significantly greater recovery rate, and proliferation capacity of germ cells compared to control. As a results, cryopreservation using tissue freezing methods in the presence of 200 mM trehalose is an efficient cryopreservation protocol for bovine SSCs. Collectively, these findings can serve as a model for comprehensively understanding the biology of SSCs and the factors that regulate male fertility. Furthermore, results of this study will be integral for the continued refinement of techniques to manipulate bovine SSCs.
