A Yunnan pony ear marginal tissue fibroblast cell line (NYPEM 2/2) was successfully established using the explant of the ear marginal tissue and then trypsinization the cells from the outgrowth. Observations on cell morphology and dynamic growth, analysis of karyotype and isoenzymes of lactate dehydrogenase and malate dehydrogenase were carried out. The expression of recombinant green fluorescence protein in the cells were also undertaken. The results showed that the population doubling time (PDT) of the cells was 24 h; the frequency of cell chromosome number to be 2n=64 was 92.9%; the banding patterns of the isozymes of the two enzymes had significant difference between the Yunnan pony ear marginal fibroblast cell line and the fibroblast cell lines of PEM 2/2, MSHEM 2/2 and BLCHE 2/2 derived from the Picdmont bovine ear, Mongolian ovine ear and Beijing local chicken embryo respectively. Tests for the contamination from bacteria, fungi or mycoplasma were negative; the transfection efficiency for the recombinant plasmid was 32.3%. This newly established cell line make the Yunnan pony breed, a national important genetic resource preserved at cell level, as well as will provide an effective experimental material for genetic studies on the Yunnan pony.
Taking Small Tail Han Sheeps ear margin as the study materials, we succeeded in establishing a fibroblast cell bank which content of samples was 52 by the method of explant culture directly and cryopreservation technique. The biological analysis results showed that population doubling time (PDT) of cells was approximately 24 h, diploid account for 90%～96% in chromosome. Confirmed by isoenzyme analysis,this cell line had no cross-contamination.The test results of the bacterium, fungi, virus, mycoplasma were negative. The transfection rate was high, and the extro-gene can be effectively expressed. Every indexes of the cell bank met all the standard quality controls of ATCC. It had not only preserved the germplasm resources of this important Small Tail Han Sheep on the cell level but also provided valuable material for the research of genome, postgenome and somacloning. Meanwhile, the setting-up of this technological platform will offer technology and theory for storing the cell genetic resources of other animals and poultry.
