Isolation And Culture Of Mouse Primordial Germ Cell

The major object of this paper is to isolate and culture mouse embryonic germ (EG) cells derived from primordial germ cells (PGCs) as well as to identify the EG cells from various aspects.Some factors influencing the efficiency of the isolation and culture of mouse EG cells have been discussed.Our work ay be useful to further study the biology of mouse EG cell and establish the EG cell lines. The result obtainde were as follows:1. Isolation and culture of mouse primordial germ cells was studied in 7.5-14.5dpc fetuses.One EG cell line was maintained the undifferentiated stste for 8 passages.2. With "several times digest",experiment was performed to compare the effec of different methods on isolation of mouse EG cells, the result suggests the three methods 0.125% trypsin+0.02% EDTA /0.25% trypsin + 0.04% EDTA and 0.05% trypsin + 0.008% EDTA are all so good as to can be used in the isolation and culture of mouse PGCs.3. When mouse primordial germ cells were cultured in three different cell medium, all can clone primary EG cells. Rat fetus cardiomyocyte cells condition medium is as good as EG cell medium with 1000IU/mLLIF,both are better than the medium contained no cytokine,it showed that rat fetus cardiomyocyte cell conditioned medium is fit for the isolation and culture of mouse PGCs4. Comparing the effect of different methods on the subculture of mouse EG cells,the method of Passage by hand have improved passage efficiency of EG cells compared to digestion together with fibroblast. the"digest-continuous dispersal"is more efficient for the work of EG cells late passages.5. Four isolation methods: co-culture back 1/3 embryonic tissue, co-culture the genital ridges, different speed attaching and puncture the genital ridges were used to improve the efficiency establishing the EG cells line from KunMing mice. Two passages ways were used to clone EG cells. It was found that co-culture the genital ridges and different speed attaching were more effective methods.6. When isolated EG cells were examined by observing morphology, AKP stain and in vitro differentiation capacity, it have a series of characters of mice EG cells.