Derivation Of ES Cell Line From 615 Mice And Differentiation Of ES Cells To Neural Cells By Induction

ES cells are pluripotent cells derived from pre - implanted embryos. With differentiation inhibitors, they may proliferate steadily without losing their undif-ferentiated state, just like other cell lines. They have three characteristics; 1. they have developmental totipotency, 2. they have germline transmission ability, 3. they can be manipulated genetically without losing their characteristics. Because of all these characteristics, ES cells have become very useful tool in life science research, especially in embryogenesis, cell differentiation, gene - modified animals and tissue transplantation. By now, ES cell lines from mice, hamsters, catties, sheep, pigs, monkeys and even human beings have been established successfully. But only ES cell lines from mice have been proved to have the germline transmission ablility and are used more frequently than other ones. So, derivation of ES cell lines from mice or applying ES cell lines of mice is one of the hot spots in life science research.Most of ES cell lines of mice are derived from 129 strain mice. 129 mice is very particular from other mice, so, it is very difficult to exclude the influence of genetic backgroud of 129 mice in genetic research. Karyotype of ES cells may change after long - term culture, and their ability to make mosaic mice may decrease, too. So, it is very important to establish ES cell lines from other strain mice and freeze them in early passages. There are no ES cell lines ready for sale established by native scientists by now. 615 strain mice are cultured by our n-ative scientists, they are easy to suffer from lung adenocarcinoma and sensitive to 638 leukemia virus. They are strong and easy to proliferate, they are very widely used in oncology, especially in leukemia research. So it is very important to establish ES cell lines from 615 strain mice.There are many ways to establish ES cell lines, we compared the efficiency of PMEF feeder layer, NIH3T3 feeder layer and LJF to seperate ES cell coloniesfrom Kunming mice. The result indicates that it is a more efficient way to separate ES cell colonies from E3 blastocysts of Kunming mice by PMEF or NIH3T3 feeder layer. Then we tried to separate ES cell colonies from 615 mice blastocysts by PMEF feeder layer and succeeded. We also suceeded in culturing 615 ES cell colonies by BRL conditioned medium. ALP staining revealed that the ES cell colonies of 615 mice are stained purple. The karyotype of these cells is 40, XY. The ES cell colonies of 615 mice can differentiate to epithelium -like, en-dothelium - like and neuron - like cells spontaneously. But there are still more examination should be done.In 1998, Thomson, et al established ES cell line from human beings which provide a better tool for human embryogenesis , cell differentiation and tissue transplantation research. The research on inducing ES cell of mice to differentiate to specific type cells may provide some useful data for relative research by human ES cells. There are two main ways to induce ES cells to differentiate to neural cells: one is ATRA induction way, the other is growth factor - mediated lineage selection way. ATRA induction way is relatively simple, but the efficiency of differentiation is very low. The growth factor - mediated lineage selection way is quite complicated, but the efficiency of differentiation is quite high. We tried to modify the traditional ATRA induction way to increase the efficiency of differentiation. The protocol is: culturing ES cells in suspension to form EB, inducing the EB by ATRA to get the mid - differentiated cells, stimulating the mid - differentiated cells to proliferate by bFGF, culturing these cells without bFGF for 7 days, then the differentiated cells showed typical neural cells morphology. It was also proved by Western blot and RT - PCR methods that the differentiated cells expressed neural relative genes and proteins. So, it indicates that the modified ATRA induction way can induce ES cells to differentiate to neural cells. The efficiency of this modified way is much higher tha...