Transgene Sperm And Cryopreservation By Transfection Of Spermatogonial Stem Cells From Cynomolgus Macaque(Macaca Fascicularis)

Appropriate animal models are essential for human disease research and drug development.Cynomolgus macaque(Macaca fascicularis)is one of the most ideal animal models for biomedical research due to the capacity to imitate etiology and development of human diseases.Spermatogonial stem cell(SSC)is one type of adult stem cell with self-renewal and differentiation potential located in the basement membrane of testicular tissue.Therefore,gene editing on cynomolgus macaque spermatogonial stem cells can be an effective approach to generate non-human primate disease models.The purposes of this study are: 1)in vivo inject enhanced green fluorescent protein(EGFP)lentiviral vector into cynomoguls testicles and transfect into spermatogonial stem cells to obtain transgenic sperm;2)or in vitro transfect the isolated spermacogonial stem cells with lentivirus vector of EGFP,and inject the spermatogonial stem cells carrying EGFP into the recipient testicle of cynomolgus macaque to obtain transgenic sperm.3)establish and optimize an egg yolk free cryopreserving system for cynomolgus sperm.The results showed that the transgenic sperm was successfully obtained that confirmed by PCR detection and immunohistochemical identification.The spermatogonial stem cells were isolated and cultured in vitro,and then the EGFP-carrying spermatogonial stem cells were successfully injected into the testicles of male cynomolgus monkey which was modeled by busulfan-treated.The addition of 0.1,1 and 10 ?g/m L of antifreeze protein ?(AFP ?)to egg yolk free sperm freezing medium can significantly improve the cryosurvival of frozen sperm.The concentration of AFP ? at 0.1 ?g/m L showed the best cryoprotection on sperm mitochondrial membrane potential.This study made a preliminary discussion on the culture of spermatogonial stem cells and the preparation of gene editing animal model based on spermatogonial stem cells,hoping to provide a reference for the study of spermatogonial stem cells.