Transfection Of EGFP Gene By Intratestis Injection And Its Expressionin In Spermatozoa

Background and purpose: Preparation of the current laboratory methods of transgenic animals is still commonly use pronuclear injection. Pronuclear injection, however need to use expensive micro-injection, higher technical requirements. In addition, some animals, particularly in dogs and other animals as a result of a large number of lipid-rich cytoplasm, it is difficult to see under the microscope prokaryotic. So that the technology to some extent has been limited. Therefore, we object to the male animals, optimize a novel direct injection of testicular, Through the spermatogonial stem cell-mediated preparation of transgenic. The production of transgenic animals is a new channel, especially for those who have not been able to manually control the breeding activities of the transgenic animal .Methods: Through the exploration of different ages, different methods and different injection volume of injected mice transfected with the impact of reproductive capacity, selected the most suitable age and injection volume of mice for research. observe the EGFP gene integration by frozen section and PCR detection.We take Beagle dogs for study, to compare the impact of different doses of transfected DNA to semen quality and testicular tissue,selected the most appropriate dose. Testicular injection study the feasibility of the direct method, and optimize the injection of testis.Results: After the injection of 5-week-old and 7-week-old mice, found that 7-week-old mice survival, breeding rate and average litter size were superior to 5-week-old mice,This experiment show that 7-week-old mice must be the best age for choice. In this experiment ,one testiculus of the mice are ligated but not injected ,with the other one injected . from which we know the reproductive capacity of the mice, ligated and injected in one testiculus, is better than that of the mice which are not ligated but injected in both testiculus. Explore the use of dye to 40μl and 60μl, 60μl found dye transfer, so that the pressure within the testis has become a lot to dye outflow from the injection hole, but also caused a certain degree of testicular damage, it's survival, breeding rate and litter size were lower than the average dose 40μl. Observed under fluorescence microscope,show that the production of frozen sections of testes of mice spermatogonial stem cells are EGFP gene expression after injection.Ten male beagle dogs, 2-3 years old and with strong sexual desire, are selected and divided into 5 groups, 2 each group.(one for semen collection, and the other is used only for the production of frozen section), non-ligated side of the testis were injected 200μg, 300μg, 400μg, 500μg, 600μg five different doses of DNA, one injection every four days, a total of three injections. 50, 60, 70,80-day collection of fresh sperm, showed that 500μg, 600μg dose group to deal with the former than the average dog semen ejaculation volume, density, color and vitality and decrease the appearance of more obvious. Observed under fluorescence microscope,the result only in the injected dose 400μg plasmid dogs (W219), in 60-70 days after injection of fresh sperm collected have weak expression of EGFP, and the remaining samples were no EGFP expression.500μg and 600μg dose have a certain amount of testicular tissue injury compared to normal dogs after 30 days injection,all the seminiferous tubules were scattered.Observed under fluorescence microscope,show that the production of frozen sections of testes of Beagle dog spermatogonial stem cells are EGFP gene expression after injection.Conclusion: In this experiment, we studied the the feasibility of testis-mediated gene transfer and optimized the testis injection. Observed under fluorescence microscope, found the expression of EGFP gene in mouse spermatogonial stem cells , dogs spermatogonial stem cells and sperm.Method to be further optimized, is expected to receive a large number of transfected EGFP gene in sperm and the sperm-derived stem cells by transplantation or in vitro intracytoplasmic sperm microinjection single transgenic animal technology The results of this experiment, we prepared transgenic dog in the road to explore a big step forward.