Research On The Establishment And Characterizing Of DaGu Chicken And QingKe Chicken Fibroblast Cell Lines

Embryos of DaGu chicken and QingKe chicken were cultivated by direct adherent culture methods to culture fibroblast. And then the cell lines were frozen in liquid nitrogen to conservation. The cells were passaged no more than 4 generations,and the cell freezing density is about 2～3×106 cell/ml, DaGu chicken and QingKe chicken's sample numbers of each kind are 19 and 9, conserved numbers are 91 and 42 ampules,biological characters of fibroblast were analysed according to the request of constructing cell line. The results were as follows:1.Cultured cells'morpha is typical fibroblast,which is fusiformate or unregular triangle, cells is radiant,flaming or rotative during growing . Through microscope the morpha diversity of fibroblast in different breed can be observed.2.The growth curve of culture cell in vitro appeared"S"shape and illustrated that cells growing included latent phase, logarithmic growth phase and stagnate phase.3.The result of cell survival rate descending showed that cells were injured during cryopreservation ,but it's survival ratio surpassed 90 %. Adherent cell after revival growed as fastly as cell before freezing. Type of cell is absolute fibroblast.4 . The result of microbial detection showed that bacteria, fungi, virus and mycoplasma did not contaminate cultured cells.5.The result of chromosome analysis: the chromosome diploid number of DaGu chicken and QingKe chicken is 2n=78, The result showed cells still kept diploid condition and the hereditary character is stable. There is no crossed contamination between cell lines.6.The results indicate that lipofectin could effectively transfer pECFP-N1 plasmid into fibroblast,for which the optimizaton transfection conditions involve in fibroblast growth state,DNA/lipofectin mixing concentration ratio,and exposure time of fibroblast to DNA lipofectin complexes. transfection efficiency of DaGu chicken and QingKe chicken were28%and32%.7.Through analyzing two kinds of isoenzyme, MDH and LDH, different breeds has specificbrands. There is no crossed contamination between cell lines. The cell lines that constructed in this study accord with the request of ATCC. We successfully constructed seven kinds of livestock and poultry breeds'fibroblast lines and conserve genetic resource...