Black Bear Somatic Cells Culture And The Bear-cattle Clone Embryo Fusion Condition Initially Searches

Bear's gallbladder, the meat, the bone, the skin and so on may be used as medicine. So bear with the ornamental value In addition, there is a higher economic value, but also precisely because it has a high economic value,leading to a sharp decline in population size.Have been classified as Category II national key protected wild animals.The black bear, the alias black bear, the black bear, distribute in a big way in Northeast, Xiao Xing'an range, Changbai Mountain, Animalia→Chordata→Vertebrata→Mammalia→Tberia→Carnivora→Ursidae→U.thibetanus。In recent years overcut seriously, once distributed very broad bears the habitat to turn the isolated plot day by day, caused the population the separation, especially Changbai Mountain area habitat vanishing was most serious, the black bear gene and the idioplasm preserved seed imminently.This study is divided into two parts:the first part of the research is create the black bear ear fibroblast cell lines by fragment of tissue with enzyme digestion method. Including the primary culture, cell purification, culture medium selection, growth curve drawing, etc.In the second research, we use Black bear-ear skin's fibroblast cells as donor cells and use MⅡenucleated oocytes as recipient cells. We do the experiment by means of acupressure enucleated oocytes putting donor cells into yolk space and use electric fused. We investigated different cell fusion mediums, different methods of fusion and suitable conditions of fusion. We discussed the best conditions of fusion for somatic cell cloning in Black bear. The result is evaluated as fused rate, cleavage rate and the development rate of reconstructed embryos in vitro. They are shown that:1.Black bear-ear skin fibroblast cells were derived by fragment of tissue with enzyme digestion method.2.The third generation of the purified fibroblast cells doubling experiments, according to cell density before and after the change, by calculating the results for black bears to determine fibroblast cell line passaged in culture medium serum concentration of 10%.3.Through the propagate regulation of Black bear-ear skin fibroblast cells was researched by drawing growth curve. It shown that the cells cultured by DMEM were in lag phase about one day, the cells come into logarithm growth about six days continuely. The cells take contact restrain and begin death, and come into flat time about three days, cells fell off and died gradually, the amout was descend.4.Electric field strength in fusion with 1100V/cm,the development rate of 16-cells is 34.89%,respectively,it is significantly higher than the other three groups (P<0.05)5.2 times of fusion pulse frequency,the development rate of 8-cells is 61.31% .it is significantly higher than the other two groups (P<0.05)6.40 us of fusion pulse duration, the development rate of 4-cells is 75%,it is significantly higher than 10μs;the development rate of 8-cells 53.89%,it is significantly higher than 20us (P<0.05) Conclusion:The results were showed that suitable Bear-cattle reconstructed embryos fusion conditions including 1100 V/cm of fusion electric field intensity; 2 times of fusion pulse frequency and 40 us of fusion pulse duration.