Effect Of Cryopreservation On YanBian Yellow Cattle Somatic Cells Culture And Efficiency Of Nuclear Transfer

The present experiment studied freezing and preserving fibroblast cells by liquid nitrogen,and made fibroblast cells as nuclear donor.Based on the culture and freeze of YanBian Yellow Cattle-ear skin fibroblast cells,used tissue culture with enzyme digestion method to establish YanBian Cattle-ear skin fibroblast cell line.I freezed fibroblast cells and observed the growth situation,get the best freezing and preserving system.And also studied the effect of nuclear transfer using fibroblast cells after freezing.The result as follows:1.YanBian Yellow Cattle-ear skin fibroblast cells were derived by fragment of tissue with enzyme digestion method.It could be derived and cultured normal cells by succeeding getting cells from refrigerate YanBian Yellow Cattle-ear skin.Compared with fresh tissue,the time of cells free and anchorage dependent was increased,living tissue was decreased.2.The result of cell survival rate descending showed that cells survival ratio surpassed 76%.After 2～3passages,adherent cell after revival growed as fastly as cells before freezing.Type of cell is absolute fibroblast.3.The fibroblasts cells of YanBian Yellow Cattle were frozen in DMEM with serum and antibiotics by adding ethylene,DMSO,glycerol.The treatment with 15%glycerol had the best effect,which can meet the needs of fistoblast passage and long-term preservation.4.The Propagate regulation of YanBian Yellow Cattle-ear skin fibroblast cells showed that cells after revival cultured by low-sugar DMEM were in lag phase in first two day,then entered log phase and persised six days,it reached peak at seven day,and began to die because of contact repression,they stayed at platform phase for about three days.5.YanBian Yellow Cattle-ear skin fibroblast cells contained normal chromosome after frozen.6.In somatic cell cloning,serum starvation and contact repression were studied as handle method of donor cells.The results showed that the cleavage ratio was different and blastocysts ratio was different(P＜0.05). So,serum starvation was the better method. 7.Injected fresh and frozen fibroblast cells with 5～10 passages and 11～15passages,the results showed that the cleavage ratio fresh group was higher than frozen group(P＜0.05);blastocysts ratio was also higher than frozen group(P＜0.05).