Establishment Of Embryonic Stem Cell Line And Transgenic Mouse Derived From Kunming White Mouse

Embryonic stem cell (ESC) is a kind of undifferentiated cell population with pluripotent differentiation potential, which is derived from inner cell mass (ICM) of mammal's blastula or primordial germ cells (PGCs). Since ESC lines were first established, ESCs have been attracting scientists'attention because of its unique pluripotency and indefinite self-renewal. Researchers hope that ESCs can provide a way to solve the current problems.The goals of our experiments are to isolate and culture ESCs from ICM of Kunming white mouse, to explore the culture conditions for Kunming white mouse ESCs, to establish the ES cell lines derived from Kunming white mouse and to generate fluorescent protein-transgenic mouse model based on the ES cell lines. Serum-free and feeder-free culture of ESCs is a difficult work, and most of ES cell lines were generated using in-bred mice of foreign strains. As out-bred mice used most frequently for scientific research, Kunming white mouse have many advantages: high reproduction, large genetic variation and low cost in scientific study, contributing to its promising prospective. The establishment of ES cell lines and transgenic mouse model derived from Kunming white mouse will facilitate the experiments in which Kunming white mouse is selected as material and reduce the costs. Moreover, serum-free and feeder-free culture could avoid the substrate from non-human animals, simplify the protocol and promoter the efficiency.In this study, we successfully obtained three strains of Kunming white mouse -derived ES cell lines through the culture of Kunming white mouse ICM, followed by many identifications. The conclusions are as follows:1. Blastula could be successfully cultured with 3i media, and ES cells were obtained from blastula.2. Kunming white mouse ES cells were successfully cultured with 2*3i media and passages exceeded 40 times.3. Kunming white mouse ES cells was shown to be response-positive in AP detection.4. Kunming white mouse ES cells was shown to be response-positive in immunofluorescent staining. 5. Kunming white mouse ES cells was shown to express Oct4 and Sox2 by Western blot.6. Kunming white mouse ES cells was shown to express Oct4, Sox2 and Klf4 on a level close to mESC and significantly different from MEF by quantitative Real-Time PCR.7. Kunming white mouse ES cells was demonstrated to form tumors in 85% of nude mice after 6 weeks.8. Kunming white mouse ES cells with red fluorescence were generated through infection of lentiviral vector, pLV300-H794.In this experiment, we used 3i media-based culture system to successfully isolate and culture Kunming white mouse ES cell lines, which had been identified through many techniques. Kunming white mouse ES cells with red fluorescence were obtained through infection of red fluorescent protein-expressing lentiviral vectors, establishing the basis for further generation of transgenic mice model. In addition, culture of Kunming white mouse ES cells with serum-free and feeder-free media supports the feasibility of 3i-based culture system.