Establishment Of Fibroblast Cell Bank For Tianzhu White Yak And Minxian Black Fur Sheep

Tianzhu white yak and Minxian black fur sheep were selected as the objects of the study, as they are the excellent varieties from Gansu province .Using trypsin heat digestion, fibroblasts cell bank from the two animals'tissues were successfully constructed. Their growth dynamic, population doubling time, karyotype analysis, isozyme pattern, microorganism contamination, transformation, expression of foreign genes and other characteristics were studied . The study aimed to successfully establish fibroblasts banks for the preservation and effective utilization of genetic resources of local ruminant species at the cellular level, and also to demonstrate the feasibility of methods for the preservation of genetic resources from other animals. The bank was also built for research on embryonic stem cells, transgenic engineering and cloning. Samples were obtained from 28 Tianzhu white yaks, resulting in the preservation of 472 frozen cells lines; from 23 Minxian black fur sheep, 290 cell lines were derived. The results are as follows:1. The cultured cells had typical fibroblast morphology, population doubling fast, grown good , their shape like irregular triangle or shuttle, appearing as radioactive blaze and helix.2. Their growth curve was"S-shaped"and showed a population doubling time (PDT) of 24 hours. After the second day, the cells went through latent phase, logarithmic growth phase and stagnant phases.3. Viability was maintained at above 90% and when frozen cells were thawed and cultured, their cellular characteristics and growth rate was not altered, demonstrating that the lines were stable"4. The fibroblasts were tested for microbial contamination and were negative ,which demonstrated that the cultured cells were not contaminated by bacteria, fungi (including yeast), viruses or mycoplasmas. 5. The number of chromosomes in the cultured fibroblast cells from the white yak were 2n=60, comprising 29 pairs of enchromosomes and 1 pair of heterosomes; the 29 pairs of enchromosome were the telocentric chromosomes, the heterosome X and the Y chromosome are the submetacentric chromosome. When chromosome numbers were analysed in 50 cells, 74% were diploid. In the Minxian black fur sheep in vitro cultured fibroblast cells, the normal chromosome number was 2n=54 , including 26 pairs of enchromosomes and 1 pair of heterosomes; 3 pairs of the enchromosomes were the submetacentric chromosomes and 23 pairs were telocentric chromosomes. When counts were performed on 50 cells, 84% showed a stable diploid number.6. Electrophoretic analysis of isoenzyme banding patterns were as follows: there were 5 bands of LDH in the cells of Tianzhu white yak, the rate of relative migration was identical, the relative migration sequences were LDH1, LDH2, LDH3LDH4,LDH5,,from"+"to"-";and there were 3 bands of LDH in the cells of Minxian black fur sheep , the relative migration sequences were LDH1, LDH2, LDH3, the results show the content of LDH in different species are different ;but the LDH zymogram in theae cells from different tissue of Tianzhu white yak or Minxian black fur sheep were identical, this shows cell genetics are stable.7.The transformation and expression of red fluorescent plasmid in fibroblast cells, this shows fibroblast cell did not cross contaminated and malignant transformed with other cell of other species ,and they transformed and expressed foreign gene efficiently. Transfection efficient could be the refers of cytoactive, meanwhile the cell of successfully transfected could provide donor cell on research of structural genomics, post genomics,transgene. By constructing animal cell banks, we can permanently conserve the genetic resources of domestic animals at cellular level, and provide valuable materials for other biological studies.