The Isolation And Culture Of Bovine And Goat Embryonic Germ Cells

The major object of this paper is to isolate and culture bovine and goat 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 bovine and goat EG cells have been discussed. Our work may be useful to further establish bovine and goat EG cell lines. The results obtained were as follows:Isolation and culture of bovine primordial germ cells was studied in 49 fetuses .We found that the bovine fetuses with the age from 29 to 50 days can be used to isolate the primordial germ cells, and one EG cell line maintained undifferentiation for 7 passages.The primordial germ cells were isolated from 46 goat fetuses embryonic age from 25 to 38 days, there were so many colonies in primary culture. One EG cell line maintained undifferentiation for 6 passages.When bovine primordial germ cells were cultured on different feeder cells: MEF,STO and BEF, all can sustain bovine primary embryonic germ cells, and MEF was the best.When goat primordial germ cells were cultured on different feeder cells, MEF,GEF and BEF all can sustain goat primary embryonic germ cells, and MEF was finer than others, but there were no significant difference between MEF and GEF(P>0.05).When the goat primordial germ cell and somatic cell were co-cultured, there are some colonies in primary culture, because the somatic cell can maintain the survival of the PGCs.Goat primordial germ cells were cultured with the medium contained different concentration of LIF, there was no difference in primary culture (P>0.05); and when passaged, the group contain 10 ng/mL LIF was finer than others, but there was no difference with the the group contain 5 ng/mL LIF (P>0.05). The group contain 1ng/mL LIF was the worst.Another experiment was performed to compare the effect of different methods of disruption on goat EG isolation. The dispersal effect of 0.125% trypsinase + 0.02% EDTA (disposed 20~30 min) was finer than that of 0.25% trypsinase + 0.04% EDTA (disposed 15 min) at 37℃.With 0.125% trypsinase + 0.02% EDTA disrupt gonad, comparing effect of different dispersal methods on the passage of goat EG cells, the method of "digest + blowing disperse" was fine, which was simple and can save more time and labours and better to keep the activity of the EG cells. One identification of PGC-like cell were accomplished by alkaline phosphatase(AKP) staining, and AKP-positive cells and colonies were found. An Immunohistochemistry assay was carried out to characterize the colonies of bovine primordial germ cells, and expression of stage-specific embryonic antigen 1, 3, 4 was detected, but the expression was feeble.