Cloned Cattle Embryonic Stem Cells, Isolated From The Primordial Germ Cells

Primordial germ cells(PGCs) were isolated from 4~13 week old bovine fetus.Some of them were cultured on mitotically inactive feeder layer cells,the others and bovine embryonic fibroblast obtained from a conceptus of equivalent gestational age were cocultured. The culture system were composed of DMEM (high glucose) supplemented with NBS, o.1mM beta-mercaptoethanol, cytokines. The purpose of this study were to obtain bovine embryonic stem (ES) cells. The results attained were as follows:1.The number of bovine fetus collected from slaughterhouse was 50. In these fetus,10 ones were contaminated during manipulation .The ES cells derived from a 6.5cm length were continuously passaged (passage 15),but 11 fetus weren't acquired ES cells. The formation rate of bovine ES cells in respective passage was: 72.5%；47.5%；42.5%；30%；22.5%；17.5%;17.5%；12.5%；12.5%；10%；7.5%；5%；5%；5%；2.5%；2.5%。 2.The growth behavior of bovine ES cells derived from PGCs was observed. A small clony composed of 4~5 PGCs was discovered after the PGCs were cultured 12h.. At the 7th day ,some nest-like ES cells clonies were observed. Primordial clonies of hill-like, steamed bread-like were black or brown, which maybe due to relatively excessive mixed cells.With the increasement of cloning passage, the number of classical nest-like ES cells clonies relatively manifolded.3.The density of new bovine serum (NBS) was influential on isolating and cloning bovine ES cells. Respective NBS density designed in this study was: 0%；5%；10%；15%；20%。The result indicated: bovine ES cells in presence of 15% NBS was easiest to isolate and clone .4.The supplemented cytokines influence on isolation of ES cells and growth of homologous bovine fetus fibroblast wasn't remarkable. Nevertheless,the influence was prominent in the process of cloning. As a result, it was best in presence of LIF, SCF and bFGF. ES cells were continuously cultured for 7.0 passage when we appended multiple cytokines; the contrast were cultured for 4.5 passage. The respective dose of LIF, SCF and bFGF was: 1ng/ml；5ng/ml；10ng/ml。5. In this research, cell dispersed liquid planed was:0.2%trypsin; 0.4% trypsin；0.04%EDTA；0.08%EDTA；0.2% trypsin +0.04%EDTA；0.4% trypsin +0.08%EDTA。We drawed a conclusion:the 5th project was relative gentle to cells and the capability of cloning was better.6.During the course of isolating and cloning ES cells, the manner of bovine ES cells and homologous fetus fibroblast coculture was dominant than the one of ES cells cultured depended on mitotically inactive feed layer. The layer cells was provided by bovine muscle or spermary embryonic fibroblast, mouse embryonic fibroblast and human embryonic fibroblast.7.In this study, the bovine ES cells continued to be alkaline phosphatase positive and had the capacity of forming embryoid bodies. Furthermore, they could sustain the development of recombined embryos and found to be karyotypically normal and stable. The cells were ES-like cells.