| China, as one of the largest animal producers in the world, is rich in animal genetic resources, and there are many local breeds scattered throughout the country. Nevertheless, current status for conservation of domestic animal diversity is not optimistic. Higher lever of industrialization is realized through raising a high-productive breed(s) or hybrid(s) to replace local breeds. Decline and deprivation of genetic diversity in poultry and livestock breeds took place progressively. Yunnan pony and Beijing yellow chicken, which are two important local breeds in China, were sampled and fibroblast cell lines of them were established and characterized respectively in this research:1) The ear marginal tissues of the Yunnan ponies were successfully cultured either by primary explant technique or by crude collagenase technique. Fibroblast cells were separated by trypsinization and three times subculturing. Morphology and analysis of dynamic growth showed that there were no significant morphological difference between cells cultured with the two techniques after three passages, and the population doubling time (PDT) of cells made with the two techniques were 36.5 h and 31.5h respectively. Therefore, it was suggested that well combination of the two techniques was an efficient and fast method for the thin tissue culture, such as ear marginal tissue of the Yunnan pony and the other animals. Moreover, it was significant for the conservation of national important domestic animals and poultry breeds being in danger of extinct at the cellular level. Otherwise, a Beijing yellow chicken embryo fibroblast cell line was successfully established using the primary explant technique and population doubling time of the cells was 39.7h. A certain extent number of cells for these two breeds were freezed, and viability examination of these cells showed that viable rates were higher than 90% before freezing and after recovery.2) Observations on morphology, analysis of karyotype, isoenzymes of lactate dehydrogenase and malate dehydrogenase for the two newly established cell lines were undertaken, and expression of recombinant green fluorescence protein in the cells were carried out. The results showed that for Yunnan pony, the frequency of cell chromosome number to be 2n = 64 was 92.9%; for Beijing yellow chicken, diploid cells were dominant of 76% ~ 88%. The banding patterns of the isozymes of the two enzymes had significant difference among the two newly established cell lines and other cell lines in our laboratory; tests for microbial contamination from bacteria, fungi or mycoplasma were negative; transfer efficiency for the plasmid was high of 33% and 25% respectively. The two newly established cell lines make the Yunnan pony and Beijing yellow chicken breeds, two national important genetic resources preserved in cell level, as well as will provide an ideal experimental material for the genetic studies on these two local breeds.3) The PrP~c genes of Yunnan pony and Beijing Yellow chicken were amplified by PCR, and the sequences were compared with those published of some species using molecular biological softwares. The results revealed that the PrP~c of Yunnan pony was remote to bovine PrP~c, therefore, the infectionrisk by cattle-derived prions might be small, and the bird PrP~c belonged to a different phylogenetic branch compared with mammal, which maybe suggest bird PrP~c can not infect mammal or can not induce protein conformation change. Thus, these results supported species barrier mechanism in the transmissible spongiform encephalopathy disease, and provided significant value for the research on functional and biological evolution of this gene.
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