Production Of The Vascular Endothelial Cell Specific Expression Of Inducible Tie2-CreER^TM Transgenic Mice

Transgenic mice were the earliest transgenic animals. This technology is developing fast in the life science for the past few years. It is already be the necessary research tool for the modern biologist. We can direct observing expressive product of the gene and the phenotypic effect though manipulate with the gene in vivo. This technology is used widespread in molecular biology, immunology, developmental biology and livestock breeding. It has a very important role in the life science research.The key point in making a transgenic animal is transferring gene successfully into the mouse eggs. There are more than ten methods to make transgenic mouse according to the different gene transfer method. A method for microinjection of mouse zygotes to produce transgenic mice is the commonly used and effectively transfered method in present. Its elementary process is to push the injection pipette which with 1μm diameter into the pronucleus of eggs -7- and inject the exogenous gene DNA solution into it. Then transfer the injected eggs into the oviduct of acceptor female mouse. The pups were born which take along the exogenous gene are the transgenic mice.This method is the effective way to make transgenic mice. To analyze gene in vivo quickly and precisely, we have set up a mouse fertilized zygote microinjection system. Including set up the lab equipment, vasectomizing male mouse, inducing superovulation, making pseudopregnancy female mouse, microinjecting and oviduct transplanting.In addition, injection into the mouse pronucleus of fertilized zygote is the classic transgenic technology. It is still the main method to transgenic animal till now. But this technology has many steps and requires operation with highly attention. To improve stability of gene expression and integration efficiency we research and optimizate many aspects of the technology.We found that IP 3~5 weeks old, body weight 12~14g CBAXC57BL/6 F1 hybrid mouse 5 IU PMSG at 13:30 in the first day, IP5IU hCG at 12:30 in the third day, then mated with stud male mouse immediately. We get the eggs at 10:30 in the fourth day. The quantity and qualitative of the eggs were the best ones. The fertilized eggs have developmented into 2 cell stage rate is the highest when we inject 1~2μg/ml(2~4pl)linearized purified DNA solution into the male pronucleus. The number of the pups were maximum when we transferred 10~15 injected eggs into the oviduct of receptor female mouse. KM is the good receptor female mouse.We generated vascular endothelial cell specific expression of inducible Tie2-CreERTM transgenic mice. First we constructed the expression vector Tie2-CreERTM-core enhancer and Tie2-CreERTM-full enhancer. We have microinjected the linearized purified DNA solution into fertilized C57BL/6 XCBA F1 oocytes. The genotypes of all offsprings were analyzed by PCR on genomic DNA from tail biopsies. Total three founder mice carrying the Tie2-CreERTM-core enhancer and one founder carrying Tie2-CreERTM-full enhancer. The integration efficiency is 6%and 5%.The Tie2-CreERTM mouse we generated in current study is a valuable genetic tool not only for endothelial cell-specific gene targeting, but also useful for endothelial cell lineage analyses. Our lab generated the conditional knock-out mouse model of the key transcription factor RBP-J of Notch signaling RBP-J flox/flox. This model mated with Tie2-CreERTM mouse. The pups will conditional knock-out RBP-J and then block the Notch signaling in endothelial cells after tamoxifen induction. Then can reveal the angiogenesis and changing of the makers though histology and immunohistochemistry.