Construction And Biological Study Of Five Local Domestic Animal Cell Line

Menggu sheep ear skin tissue, Beijing duck embryos(14days),Qingyuan flesh chicken,Zang chicken and Langshan chicken embryos(8days), were cultivated with direct adherent culture methods to get fibroblast, then they were frozen in liquid nitrogen successfully.The cells were passaged no more than 5 generations,and the cell freezing density is about 3×106 cell/ml, sample numbers of each kind are between 30 and 60, conserved numbers are 150-180 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 .Qingyuan flesh chicken's cytoplasmic tubercle is much more and shorter in contrast with Zang chicken and Langshan chicken,that can be regarded as different character from others. 2 The growth curve of five kinds animal's 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 results of microbial detection showed that cultured cells were not contaminated by bacteria,yeasts,fungi,virus and mycoplasma. 5 Chromosome number of menggu sheep, Beijing duck and chicken is respectively2n=54, 2n=78 and 2n=78±,Results of chromosomal karyotypes indicated that cells are diploid,and the hereditary character is stable.chromosome character can identify cell lines. 6 Results of isoenzyme analysis showed that isoenzyme bands of each kinds of animals has different characters, so decluded cell line were not contaminated each other. 7 Transfecting fibroblast with lipodfectin mediated plasmid DNA This results indicate that lipofectin could effectively transfer pEGFP-N3 plasmid into fibroblast, for which the optimization transfection conditions involve in fibroblast growth state, DNA/lipofectin mixing concentration ratio,and exposure time of fibroblast to DNA lipofectin complexes. The highest efficiency was achieved with cultured fibroblast at 70% and 80% confluences .the essential transfection condttitions for these efficiencies were in coating pEGFP-N3 of 1.5μgDNA /100μl with lipofectin 2μl/100μl,and exposing the fibroblast to the DNA-lipofectin complexes for 10 hours . transfecting efficiency of menggu sheep is 52%,Beijing duck is 23%,Qingyuan flesh chicken is 33%,zang chicken is 20%,langshan chicken is 19%。By Constructing animal cell lines, not only genetic resource of domestic animal is permanently conserved at the level of cell, but also the valuble materials were provided to biological studies.