Preliminary Research Of Producing Transgenic Mouse Embryos By Lentiviral Transgenesis

Transgenic animal models have become the most appropriate experimental materials in the field of life sciences and medicine to research the gene structure and function, gene therapy, organ transplantation and the animal models of disease and in the field of Animal Science to improve animal production and so on. It is the bridge of linking the molecular level, cellular level and the level of live animal research. There are many methods to prepare transgenic animals, such as:DNA pronuclear microinjection, retrovirus-mediated transgensis, embryonic stem cell mediated, somatic cell nuclear transfer and sperm-mediated ect.. In all the methods mentioned above, lentiviral transfer is the most common method of preparation of transgenic animals. Currently, lentiviral transfer has been successfully prepared transgenic mouse, large mammals such as cattle, sheep and pigs and poultry. The efficiency of transgenic animals is much higher than traditional methods. Because of the unique physiological and genetic characteristics of the mouse, it has become the best suitable animal model in the field of life science for the research of gene function, developmental biology, and construction of animal disease models and so on. In this study, In the process of producing porcineβ-defensin-2 transgenic mouse model, we preliminary study the conditions of the KM mouse for super-ovulation, the method of embryos picking, the method of embryos in vitro and analyze the survival of the embryos after infected by lentivirus; Through exploring these conditions, we plan to establish the transgenic animals platform with the lentivirus infection, Which can lay the foundation for analyzing the gene function in vivo and constructing related diseases model of mouse to study the pathogenesis of animal diseases. This study mainly includes two parts as below:Packaging of the replication-defective lentivirus. We used the fourth generation lentiviral vector system in our study, it is constituted by the four plasmids. The PBD2 fragment was inserted to multiple cloning sites (MCS) of the pCA vector, which has the chickenβ-actin promoter (CBAP) sequence that can enhance expressed in eukaryotic, and also has the chickenβ-actin intron (CBAI) sequence and the rabbitβ-globin intron (RBAI) sequence. In this study, we transformed the PBD2 fragment which was added the CBAP, CBAI and RBAI sequence in upstream sequence to the backbone vector to construct recombinant plasmid (named:pLenti-CABD2). Then, we extracted the endotoxin-free plasmids and co-transfected 293ft cells, collected the recombinant replication-deficient lentivirus particles, the titer was 7.2×106TU/ml. The experiment that zona pellucida-free embryos infected by lentivirus in vitro showed that:when the titer exceeds the 104TU/ml. the infection rate was 100%, at the same time, we can transfer the 1-cell,2-cell and 4-cell embryos into the oviduct uterine tube of pseudopregnant mouse, and also, the 8-cell embryos can be transferred to uterus of pseudopregnant mouse, So the recombinant lentivirus can be as to prepare the transgenic mouse. These conditions provide a new way of thinking for the preparation of transgenic mouse infected by lentivirus.Super-ovulation and mouse embryos in vitro. Mammalian early embryos in vitro are the key link for the method of preparing for the transgenic animals which is co-infection by lentivirus. Super-ovulation plays a decisive role to get the embryos. In this study, we compared different doses of hormones; the results show that 10IU per experimental mouse is the optimal dose. By comparing with the method of the obtain embryos, we found that the method of combining the two was the best way to obtain 8-cell and morula embryos. Many domestic and foreign researchers have researched in-depth embryo culture medium in vitro and method of culture in vitro, until now, there are many kinds of embryo culture medium used; in our experiment, we have selected the M16 and KSOM embryo culture medium in vitro, the results show that the effect of two embryos culture medium for the embryos short-term culture was not obvious; In the aspect of methods, we compared the micro drop culture with open culture systems for effecting the cleavage of embryos and the mortality of embryos after culture in vitro 48h, the results indicated that the micro drop culture is superior to an open culture during the short-term culture in vitro.