| In order to permanently preserved the Chinese pastoral dogs’ genetic resources at the cellular level,and enable the preserved cells to prepare for the next nuclear transfer on the basis of the canine superovulation technique.1 of eight months old Chinese pastoral dog were as the research object,to built Chinese pastoral dog’s ear fibroblast cell line by tissue mass culture method.According to the specific requirements for cell lines established of ATCC,the healthy and usable cell line was established by primary culture,passage culture and detection of biological characteristics.Results:The cells cultured in this experiment showed typical morphological characteristics of fibroblasts at each stage of culture.Individual differences occurred in primary culture with different days and density,and the growth trend was radial or whirlpool.Cell viability was detected by trypan blue staining,the cell survival rate descended after cryopreservation and the viability of cells after resuscitation was lower than that before the cryopreservation,but they were kept above 95%.Using a counter to count the number of F2 F4 F6 cells in a week,the growth curves of the cells were plotted,all of them were of "S" type,which was consistent with the general law of cell growth.In DMEM containing 15%fetal bovine serum for 6 consecutive passages,there was no significant difference in cell morphology and growth.The result of microbial detection showed that bacteria,fungi,virus and mycoplasma did not contaminate cultured cells.Karyotype analysis:In vitro,the normal chromosome number of fibroblasts cultured in Chinese pastoral canine ear was 2n=78,including 38 pairs of autosomes and one pair of sex chromosomes.In the measured metaphase cells,the result showed 2n = 78 were 90%,which indicates the cell lines were diploid cells in the main and genetic performance were stability.Conclusion:the ear tissue fibroblast cell line of Chinese pastoral dog meet the requirements of cell line construction of ATCC. |
