Study Of The Transgenic Efficiency By Intratesticular Injection To Produce Transgenic Mice And Production Of Transgenic Mice Carrying Thanatin Gene

In the past 30 years, the research and application of transgenic technology in gene expression of animal had became noticeable advancement in experimental biology and applied biology. Microinjection,retrovirus mediated method and embryonic stem cells method were all the traditional methods of producing transgenic animals. But these methods had defects and limited application in the research of transgenic animals in the future. Using intratesticular injection is a new method to product transgenic animal recently. There are many merits of intratesticular injection, simple operation, low cost, higher efficiency and suitable producting transgenic animal. There are important significance to study this method for producting transgenic animal, disease-resistant animal breeding and production of valuable proteinum through transgenic animals.Two parts were included in the studies:PartⅠ: Study of the Transgenic Efficiency in Difference Spermiogenesis Stages in MiceIn this experiment exogenous gene was transferred into germ cells of male mice, in order to study efficiency of transgenic mice in different spermiogenesis stages.The green fluorescence protein expression plasmid(pIRES2-EGFP)and liposomes were mixed and injected into male mouse testis and epididymis. Then the intratesticular mice were mated with female at day 7,16,30 and 42th after infection. Polymerase chain reaction(PCR) and southern blot were applied to identify transgenic mice. The positive ratio was 6.82%,0,56.86%,42.86% by PCR analysis and 6.82%,0,47.06%,34.69% by southern blot analysis . The transgenic mice showed green fluorescence in fluorescence imager and fluorescent microscope under EGFP excitation light (488nm). Through comparing efficiency of transgenic mice in different spermiogenesis stages, it could provided an important theoretic foundation for efficient production of transgenic animals by testis mediated gene transfer.PartⅡ:Study of Transgenic Efficiency by Intratesticular Injection in Different Spermatogenic Cycles in MiceThe green fluorescence protein expression plasmid(pIRES2-EGFP)and liposomes were mixed and injected into male mouse testis. Then the intratesticular mice were mated with female at day 42,84,126,168 and 210th after injection. Polymerase chain reaction (PCR) and southern blot were applied to identify transgenic mice. The positive ratio was 6.36%,17.78%,12.50%,10.87%,4.65% by PCR analysis and 31.82%,13.33%,8.33%,6.52%,2.33% by southern blot analysis . The transgenic mice showed green fluorescence in fluorescence imager and fluorescent microscope under EGFP excitation light (488nm). Through comparing the efficiency of transgenic mice in different spermatogenic cycles, it shows that the spermatogonial stem cells could carry exogenous genes for a long time but the efficiency decreases with time. This provided an important theoretic foundation for efficient production of transgenic animals by testis mediated gene transfer.PartⅢ:Preliminary Research on Production of Transgenic Mice Carrying Thanatin GeneThanatin gene was constructed by overlap extension PCR and inserted into pIRES2-EGFP vector. Then recombinant plasmid pIRES2–EGFP -Thanatin and liposome were mixed and injected into male mouse testis. Six weeks after injection, male mice mated with female mice. Genomic DNA was extracted from F1 pups tail.Polymerase chain reaction(PCR) and southern blot were applied to identify transgenic mice. Six mice were selected from F1 positive mice, then mated with normal mice and detected by PCR. Fifty two F1 pups were born and the positive ratio was respectively 38.46% and 30.77% by PCR and southern blot analysis. In F2 pups, the positive ratio was 36.36%. The result showed that thanatin gene was integrated into mice genomic DNA. Expression of green fluorescence protein in transgenic mice was observed in fluorescence imager. In the present study, establishment of transgenic mice carrying thanatin gene is attractively valuable and prospective in application. And this provides a basis for disease-resistant animal breeding and production of antibacterial peptides through transgenic animals.