Sperm-mediated Human Coagulation Factor ¢ù Transgenic Sheep Study

Since 1989 Lavitrano described the binding of exogenous DNA in mouse, evidence has accumulated that clearly indicates that spermatozoa have the surprising property of the allowing the intemalization of molecules of different size and nature, including DNA. A large number of hDAF transgenic pigs to be used for xenotransplantation research were generated by using sperm-mediated gene transfer (SMGT). The efficiency of transgenesis obtained with SMGT was much greater than with any other method (Lavitrano et al., 2002). On the basis of the results of previous SMGT, we try to explore the prescreening parameters of sperm donor in sheep, analyze the molecular mechanism of SMGT in sheep, our study will be of benefit to those whose work requires the use of transgenic large animal models in medicine and animal biotechnology. On the first time, the study explored and established the procedure of prescreening of sperm donor in ram. Standard parameters used in conventional animal breeding programs (volume, concentration, presence of abnormal sperm cells, and high progressive motility) were used to prescreen 21 Ram, mainly focusing on motility at time of collection, high progressive motility after 2 hr. We performed an osmotic resistance test to evaluate the integrity of membrane function and assessed sperm membrane integrity by SYBR-14 and propidium iodide (SYBR-14/PI). Finally another important parameter evaluating to optimize the amount of exogenous DNA per spermatozoa, which is whether sperm is resistant to increasing concentrations of DNA, was used to select the donor of sperm. In this study, 2 of 21 rams studied were used as sperm donors. Molecular mechanism of SMGT in sheep was explored. HAM of ram sperm was studied among the SFM, TALP+Ca²⁺ , TALP free of Ca²⁺, the fate of the foreign DNA internalized in ejaculated spermatozoa in sheep was tracked. Our results showed that the sperm nucleus is not a safe place for exogenous DNA since its intemalization activates a metabolically active process similar to apoptosis which destroys both the DNA and the sperm cell. The endogenous nucleases in ram spermatozoa, which may cause degradation of DNA at hypersentive sites of sperm chromosome under certain condition (the different foreign DNA), were activated in the medium either containing Ca2+( TALP+Ca²⁺) or free of Ca²⁺ (SFM). The activation of endogenous nucleases in ram spermatozoa was associated with the sequence and conformation of the foreign DNA, EDTA failed to prevent foreign DNA-dependent chromosomal damage, the endogenous nucleases was not activated in a dose-dependent. Those results indicated that the suitable sequence of foreign DNA was important for the higher efficiency of SMGT. It is reasonable to predict that spermatozoa would have some...