Experimental Studies On Generation Of Transgenic Mice By Intratesticular Injection

A transgenic animal is one that carries a foreign gene that has been deliberately inserted into its genome.transgenic animal have extensive perspective of research and application in field of medicine, life science and biopharmacy, for this reason, scientists had been explored a more convenient and easy, economic and practice, and high-performance transgenic methods. Recently a few years, with the development of the science and technology, in vivo gene transfer have become a popular tool to be used for the fields of the gene therapy, biological analysis.Liposome is a new type of media transfection, it carries the target gene into cells by cells phagocytosis. There are many advantages of liposome, such as low toxicity to cells, easy preparation, low immunogenicity, and it can carry a high molecular DNA type, so it has more and more extensive scope of application. Not only can be used for transient transfection, can also be used for the establishment of a permanent expression.Spermatogonial stem cells are located in the membrane of the seminiferous tubules, SSCs is the only cell type in postnatal mammals which has the capability to self-renew and to contribute genetic information to the next generation. Their proliferation process can throughout the whole life. In order to investigate the feasibility of transgenic animals in injection of vivo testis, the first exploration is the spermatogonial stem cells culture conditions. Transfection Reagent is Liposome parceling of green fluorescent protein reporter gene plasmid pIRES-EGFP in different proportions. In different time points after transfection, the cells respectively observed under fluorescence microscope. The results showed that with the support of co-cultured cells can promote the survival and proliferation of SSCs. High concentration Lipofectamine2000 has toxic effect on cells, low concentration, low transfection efficiency. By cell viability counting, the best ratio of DNA and Lipofectamine 2000 is 2μg: 5μl. 36 h after transfection, cells transfected with the highest rate reached 31.5%.Based on the cells experimental, mice were injected in intratesticular. By surgery, A group mice was injected in seminiferous tubules under the stereomicroscope. Transfection Reagent mixed with 1% trypan blue which can indicate injection. Selecting 2 to 3 injection point in testicular surface can make sure 30 percent of the small-seminiferous tubules Blue. Considering the volume of the esticular, the injecting volume of each mouse is 40μl. Without surgery, B group mice were injected in intratesticular with several points and several times. The injecting volume of each mouse is 40μl per time. The results of PCR show that transgenic positive rate of A group is 31.4% in F₁ generation, 11.7% in F₂ generation. The transgenic positive rate of B group is 17.1% in F₁ generation, 12.5% in F₂ generation. Based on the development of transfection of spermatogenic cells by Lipofectamine2000 in vivo, a novel method to transfer gene was established.