A Study On The Sperm-mediated Gene Transfer Methods And The Production Of Transgenic Embryos By In Vitro Fertilization In Goats

Sperm-mediated gene transfer (SMGT) is a simplified and effective method to produce transgenic animals. Animal sperms are spontaneously capable of taking up DNA molecules, so sperms can be used as the vector to deliver exogenous DNA into animal genome in the process of animal breeding. SMGT is superior to other methods in transgenic animal production for its simplicity, lower cost and relatively higher efficiency. However, SMGT can not, at present, be used as a conventional method yet for its uncertainty and instability. To obtain higher efficiency of transgenic animal production by SMGT, it is necessary to screen transgenic embryos by biopsy prior to transfer. In vitro fertilization is an essential element of SMGT, thus optimized in vitro fertiliztrion protocol is needed to improve the effectiveness and stability of SMGT. Using occytes obtained by OPU, in vitro fertilization rate can be significantly increased. At present, there is no report focusing on systematic methodological comparative studies on sperm gene transfection protocols, and some transfection protocols (i.e. electroporation) have never been investigated in goats, except a fewer reports on transgenic goat production by SMGT is available. Therfore, in this paper, in order to establish simplified and effective technical platform for transgenic goat production, systematic intensive studies on DNA transfection of sperm and in vitro production of transgenic goat embryos were carried out.The present study is divided into two parts. The first part studies 3 methods of the exogenous gene transfection mediated by sperms, i.e. co-incubation of exogenous DNA and sperms, introduction by electroporation, and transfection mediated by liposome. The first part also investigates the factors influencing the efficiency of exogenous DNA transfection mediated by sperms, optimizes the procedures of transfection mediated by sperms, establishes a methodology of transfection mediated by sperms in goats. The second part investigates the factors of IVF and the fertilizability of oocytes obtained by OPU, optimizes IVF systems, establishes stable production systems of in-vitro embryos, studies the...