Cryopreservation And Explorer Transgene Of Chicken Spermatogonial Stem Cells

Spermatogonial stem cells (SSCs), as the originated cells of germs in male, which was immortalized and could be proliferate in the whole developmental process. Up to date, SSCs is a focus in the field of developmental biology, which has widely prospects of application in clinical of veterinarian and producing transgenic animals. In this study, the SSCs from chickens'embryos hatched for 19 days were isolated for cryopreservation, so as to search for suitable cryopresevation system for SSCs . At the same time, SSCs were transfected by exogenous DNA(pEGFP-N1) in vivo, and the expression of pEGFP-N1 were detected in sperm, embryo discs and hatching embryos in different periods, tissues, which provide the basement for producing transgenic chickens. The results were showed as following:1 The effect of Dimethylsulphoxide (DMSO) , ethylene glycol (EG) and glycerin(Gly) cryoprotectants, each at (5%, 10%, and 15%) concentration on the 19d chicken embryonic SSCs was investigated. The results indicated that (1) When the concentration of DMSO were 5%, 10% and 15%, the viability rate were 73.1%, 88.6% and 74.8% respective, and the difference were significant(P<0.05). When the concentration of EG were 5%, 10% and 15%, the viability rate were 69.4%, 83.1% and 65.2% respective, and the difference were significant(P<0.05). When the concentration of glycerin were 5%, 10% and 15%, the viability rate were less than 15%, and difference were not significant (P>0.05). (2) The SSCs, 10% DMSO as freezing media, were cultured on the chicken embryos cells as layer cells after thaw, then found the SSCs surviving well and forming colony which were positive after AKP staining, but when the SSCs were cultured without layer cells, they couldn't form colony. When the SSCs, 5%, 10% and 15% EG as freezing media, were cultured after thaw, then they could proliferate, but couldn't form colony with or without layer cells. When the SSCs, 5%, 10% and 15% glycerin as freezing media, were cultured after thaw, then they survived about 12h. The results showed that 10%DMSO were the best freezing media for the 19d chicken embryo...