The Establishment Of Quail Primordial Germ Cells LINE And Identification

Primordial germ cells(PGCs)are the precursor cells of sperm and ovum.P GCs own the potential to develop to a complete individual.PGCs can be indu ced to all kinds of cells involved 3 germ layer in vitro.PGCs have the capaci ty of racial transmission,and they can integrate with another normal embryo t o be a animal individual.In this research,we set chick fibroblast and some gr owth factors as basis and researched the separation,purification and cultivation of quail embryo.Quail PGCs cryopreservate results were also explored.1.The production of Chick fibroblast feed layer Hailan white eggs incubat ed six days were used for experimental materials.After 2-3 generations,cell m onolayers were used as feed layer after mitomycin-C treatment.2.PGCs isolation and idengtification In this experiment,we used incubated 5.5 days quail embryos to obtain cells with enzymatic digestion.We discussed their digestion time in the study,and found that the greatest number of cells were obtained after 15 minutes digest.An average of 226 cells were got in ea ch gonad.PGCs were identifiedby morphology and alkaline phosphatase(AKP).3.The subculture and culture of PGCs We cultured PGCs in the culture l iquid without feed layer and growth factors,in chicken fibroblast feed layer,a nd in chicken fibroblast feed layer which add LIF \ SCF \ bFGF.In the cultu re liquid without growth factor and feed layer,stromal cells fibrous grew adhe re to the wall and culture time was no more than 5-6 days.Cultured in chick en fibroblast feed layer,cells differentiation were inhibited effectively,remained undifferentiated individual state after 2 days.Although some cells split after 3 days,the differentiated degree was better than that without feed layer,and the living ratio was about 50%.When added growth factor to the fibroblast feed layer,there were no cells differentiated in 3 days and remained undifferentiated state.Cells began to split after 4-5 days,and ratio of the living PGCs was a bout 76%.Cell had 7-8 cell-clones when cultured for 5-6 days.When cultured to 10 days,the cell-clones reached to 13-15.The Cell morphology was not ob vious,and the blank was not close between cells.Many fibroblast were dead. We passed PGCs in the 7th day.In this experiment,PGCs could spread to 5 generation and still had the morphology of PGCs,and some could split.4.PGCs frozen and thaw Thawed PGCs preserved for two months with two methods.The ratio of living cells was 61.4%(DMSO)and 54.2%(EG+P VP). These results showed that:Enzymatic isolated PGCs in the 5.5 days gonad quail,cultured PGCs in chicken fibroblast feed layer with growth factor,passage d quail PGCs in 7th day,spread to five generations.Some of cells were still s urvival and had the breeder potential.Quail PGCs preserved two months after thawed had high survival rate and had PGCs forms.The establishment of PGC s line could provide valuable experimental materials to study development mec hanisms and the tumors for the reproductive system.PGCs also were the ideal vector of transfering target gene directly to future generations,and had temptin g prospects as Mediated Gene Transfer.