Optimizing Of Lentivirus-mediated RNAi Transgenic Goat Identification Methods And Preliminary Safety Testing Of Ovarian Injection Of Lentiviral Vector Ewes

Since Jaenisch got the integration SV40DNA mouse by injected SV40DNA into mouse blastocyst,and now,more and more people use lentiviral vector and homologous recombination to construct variety of transgenic animals. Transgenic animal identification technology aways born with the transgenic animals constructed.Although,the transgenic animal identification technology don’t develop so quickly as transgenic animals,this technology also become more convenient,quicker and cheaper.With the development of transgenic technology, more and more transgenic animals and plants emerged, GM products have the commercialization and also Appear in people’s lives from the laboratory. However, the security of genetically modified organisms, especially the security issues of genetically modified animals has been the focus of public, also whether GM products have the ability to realize the industrialization is the key of these problems. To establish the transgenic animals and their product safety evaluation system will speed up the people to accept the GM products.In this research, I used PCR, southern blot analysis, gene sequencing and routine blood test and other technology to identify lentivirus-mediated RNAi transgenic goat at the DNA level and I established the detection method of the experimental conditions less demanding. I have test ovarian injected lentivirus of safety testing of transgenic animal and proved that the exogenous gene did not transfer in ewes in vivo and lentiviral vectors didn’t impact on the health of the ewe.The research covers the following two main components:1. Identification of Lentivirus-mediated RNAi transgenic gene goat and establishment of Transgenic animal identification methods.The lentiviral RNAi vectors with the shRNA sequence of MSTN and INHA were injected into fertilized eggs of the goat by Pronuclear microinjection, and the fertilized egg after in vitro for48h, transplanted into recipient ewes, and finally we got the18lambs, extraction the Tissue DNA of lamb, I identified these lambs the BSD gene-specific lentiviral transgenic at the DNA level by PCR and Southern Blot.While the final outcome of transgenic goat born. However, I established a transgenic animal identification technology platform in the laboratory through these lamb identification of transgenic animal.And the experimental conditions and experimental procedure of Southern Blot technique has been improved by DIG HIGH Prime KIT, and I introducted the200mA power to transfer the DNA on the agarose gel transferred to nylon membrane, and proved that the power transfer could be effective after8h, and completed after12h. Without affecting the reliability and sensitivity of experiments, I used commonly used laboratory instruments such as thermostatic shaker and oven replaced the water bath shaker and the UV cross-linking hybridization instrument to complete the experiment by appropriate temperature and time regulator to reduce the experiment the requirements of the experimental conditions. So I established an experimental conditions which is a low, simple operation, high reliability and lentivirus-mediated RNAi transgenic animal identification methods.In this experiment I established the lentiviral mediated of RNAi transgenic gene goat detection technology platform, and improved the Southern Blot. Without affecting the experiment results I used the commonly used instrument to take the place of the expensive and complex equipment.Also the200mA power transfer system is introduced in DNA transfer membrane experiment, making the technology easier to operate the experiment to lower.2Safety testing of ovarian injection of lentiviral vector ewesIn this experiment, I injected0.25ml lentivirus medium on both sides of the ewes ovarian, and sent the fresh blood for the routine blood test after6month and extracted the tissue DNA from ewes blood.I identified specificBSD gene integrated into the ewes genome for the safety of injected with lentiviral vector ewes. Routine Blood test results the ewes of experimental group indicators of differences in RBC, WBC are not significant.This test proved that the lentiviral vector didn’t have effect in ewe’s healthy. Especially ewes after injection of the virus through natural mating, pregnanted success, it means there was no effect on the reproductive function of ewes ovarian injection of lentiviral vector ewes.