Stem Cells and Cloning: Advances and Applications (May 2022)
Comparative Efficiency for in vitro Transfection of Goat Undifferentiated Spermatogonia Using Lipofectamine Reagents and Electroporation
Abstract
Wilkister Nabulindo Nakami,1– 3 James Nguhiu-Mwangi,2 Ambrose Ng’eno Kipyegon,2 Moses Ogugo,1,3 Charity Muteti,1,3 Stephen Kemp1,3 1Livestock Genetics, International Livestock Research Institute, ILRI, Nairobi, Kenya; 2Department of Clinical Studies, Faculty of Veterinary Medicine, University of Nairobi, Nairobi, Kenya; 3Centre for Tropical Livestock Genetics and Health (CTLGH), ILRI, Nairobi, KenyaCorrespondence: Wilkister Nabulindo Nakami, Livestock Genetics, International Livestock Research Institute, ILRI, 30709-00100, Nairobi, Kenya, Tel +254 711 761 459, Email [email protected]; [email protected]: Spermatogonial stem cells (SSC), also referred to as undifferentiated spermatogonia, are the germline stem cells responsible for continuous spermatogenesis throughout a male’s life. They are, therefore, an ideal target for gene editing. Previously, SSC from animal testis have been isolated and transplanted to homologous recipients resulting in the successful reestablishment of donor-derived spermatogenesis.Methods: Enhanced green fluorescent protein (eGFP) gene transfection into goat SSC was evaluated using liposomal carriers and electroporation. The cells were isolated from the prepubertal Galla goats testis cultured in serum-free defined media and transfected with the eGFP gene. Green fluorescing of SSC colonies indicated transfection.Results: The use of lipofectamineTM stem reagent and lipofectamineTM 2000 carriers resulted in more SSC colonies expressing the eGFP gene (25.25% and 22.25%, respectively). Electroporation resulted in 15% ± 0.54 eGFP expressing SSC colonies. Furthermore, cell viability was higher in lipofectamine transfection (55% ± 0.21) as compared to electroporation (38% ± 0.14).Conclusion: These results indicated that lipofectamine was more effective in eGFP gene transfer into SSC. The successful transient transfection points to a possibility of transfecting transgenes into male germ cells in genetic engineering programs.Keywords: eGFP, culture, pre-pubertal
