Studies On Gene Transfer In Goat Using Spermatozoa As Vector

Transgenic animals are being developed for a variety of purposes including; models for human diseases or for use in human drug testing, value-added agricultural livestock with improved food product or disease resistance characteristics, human organ donors, and bioreactors to produce materials for industrial and biomedical applications. However production transgenic animals is very difficult and inefficient, especially in farm animals such as cow, goat ,sheep, pig and so on. Nowadays, pronuclear microinjection, retrovirus-mediated gene transfer and embryonic stem cell (ETS) transfection are the main conventional methods to introduce foreign genes in transgenic animal production. Being the most widely-used method for transgenesis, microinjection reqires costly sophisticated equipment and special experimental skills. Though successfully applied to mice, the experiment eficiency of microinjection is much lower when applied to other species, such as livestock or marine animals. The disadvantages of retrovirus-mediated gene transfer include the relatively small amount of genetic information (<10kb) that influence expression of transgene caused by the so-called "position effect", and the complexity of the process in vector preparation. Embryonic stem cell is difficult in culturing and did not established cell line except in mice and human. Therefore the seach for a simple, efficient, and widely applicable method(s) for inserting foreign genes into a variety of mammals is needed.Sperm-mediated gene transfer was established by lavitrano in 1989.She obtained 30% F1 founders of transgenic mice following in vitro fertilization of eggs with sperm incubated in the presence of plasmid DNA. The most obvious advantage of sperm-mediated gene transfer is its simplicity and the minimal embryo handling required. From then on,several reseach groups have obtained transgenic animals such as mice, rabbits, pigs, cows, sheeps, hens, fish and so on, using spermatozoa as vectors for introducing foreign DNA into embryos during fertilization.These reports proved that sperm-mediated gene transfer,an innovative method,is feasible and efficient. However others reported that no positive transgenic embryos and/or animals were obtained. These contrary results suggested that the method were necessary to reaseach, as Brinster stated, "Since the method would be of considerable value in biology, we believe it should continue to be pursued . Perhaps thenext step is for the authors to demonstrate in observing the essentuial elements of their method that have eluded us and orthers". Sperm-mediated gene transfer is based on stablized and efficient technic systems of fertilization in vitro and spermatozoa transfection. It has been proved that outstanding systems of fertilization in vitro is not only able to offer more oocytes and/or embryos, but also influence fetus development and individual health after embryos transfer. Moreover highly spermatozoa transferction rate determinates the frequency of positive transgenic embryos and/or F1 founders. Up to now, the rates of maturation, fertilization, and development of goat oocytes in vitro are low and the highly efficient systems of goat spermayozoa transfetion are not established. In order to establishe technic platform of sperm-mediated gene transfer for goat, to improve the efficiency of fertilization and spermatozoa transfection in vitro, to obtain bioreactor of mammary gland in transgenic goat, and to expore the mechanism of spermatozoa taking up exogenous DNA, we have carried out the studies on the fertilization and spermatozoa transfection of goat oocytes and spermatozoa in vitro. The main results are as following:1. Oocytes from >2mm diameter follicles on goat ovaries were better oocytes source for in vitro maturation because about seventy percentage ocytes from >2mm diameter have developed to GVâ…¡or beyond GVâ…¡.On the contrary, about 70% oocytes from <2mm diameter follicles were only developed to GVâ… . These oocytes were deficient to futher developmental competence according to...