Fibroblast Cell Culture And Heterogeneous Embryo Reconstruction In Gaint Panda

abstract Ph.D. Dissertation FIBROBLAST CELL CULTURE AND HETEROGENEOUS EMBRYO RECONSTRUCTION IN GAINT PANDA Doctoral Candidate: LI YU Advisor: Prof.: Zhang Yong The College of Animal Science and Veterinary Medicine, Northwest Science and Technology University of Agriculture and Forestry, Yangling, Shaanxi, P. R. China 712100 Abstract Experiments of generating the skin fibroblast cell line from the tissues of goat and bovine had been made in advance in order to investigate the methods of the cell line establishment and characterization. Then the finite and infinite skin fibroblast cell line from the tissue of giant panda was successfully established, and the interspecies nuclear transplant blastocysts between giant panda and bovine were obtained. Following is the results: 1. All block of skin tissue from goat can be survived in M199 supplemented with 10%NCS. 100 IU/ml penicillin. 100 lU/mi streptomycin at 4XD in 8 h. The survival rate of the skin tissue pieces in M199. DMEM. HLH supplemented with I0%NCS. 100 ILT/ml penicillin. 100 lU/mI streptomycin was 75.0%. 71.4%. 80.0% respectively. When the fibroblast cells were trypsined by D-hanks?with 0.25% trypsin . 0.02 EDTA. 0.25% trypsin桬DTA (TE) at controlled temperature and time, Attachment rate of the cells after 24 h was 86.4%. 88.5%. 95.8% respectively. According to the different sensitivity to TE, and through 1-3 passages, epithelia and fibroblast cells can be separated and purified. Cultured fibroblast cell line of Angora goat. Boar goat. Milk cow and Ji-ning goat was preserved at low temperature with the cryogen of DMSO and when they were thawed, the seeding efficiencies were 84.2%. 86.4%. 89.5% and 91.8% respectively. Thawed cells were passaged 3-5 times in culture with the natural characteristics. After thawed, the fibroblast cells of Ji-ning goat were used to study animal cloning in our laboratory, and the first adult somatic cloned goat were obtained in the world. This demonstrates that preserved cells maintain the natural genetic pattern. 2. Following the cell culture experiments of goats and bovines, we investigate the capture of skin tissue, primary culture, cell purifying, medium selection, cell preservation and the characterization of giant panda: - 97 - 2.1. The skin tissue from giant panda was brought to laboratory in M199 supplemented with 10%NCS, 100 lU/mi penicillin, 100 lU/mi streptomycin at 4C in 4 h. When cultured in the incubator at 5%C02, 95%air, 100%humidity, 37.5X2, the survival rate of the skin tissue pieces in DMEM, HLH, M199 supplemented with 10/oNCS, 100 lU/mi penicillin, 100 lU/mi streptomycin, was 75.0%, 7 1.4%, 80.0% respectively. Outgrowth of fibroblast cells started at 3-8 day from the culture beginning. The migratory efficiency of the fibrobiast cells is higher than that of the goat and cow. The configuration of the fibrobiast cells of giant panda is either different from those of goats and cows. 2.2 The fibroblast cells of giant panda can be separated and purified when trypsined with TE at p controlled tem...