Effect Of Human Telomerase Reverse Transcriptase On Lifespan Of Bovine Ear Fibroblast And Nuclear Transfer

Due to the limitation of cell lifespan, the fibroblasts begin into senescence at 50PDs and to crisis at 80PDs. It cannot be satisfied with the demand of nuclear transfer and limit the practical application in research works. In order to be sure the extra gene can be integrated and expressed normally in cells, as the donor cells for transgenic nuclear transfer, the fibroblasts should at least survive above 45PDs. This experiment transfered the plasmid which was with EGFP report gene and hTERT gene to bovine ear fibroblast through the lipofactamine 2000 and reviewed the effect on nuclear transfer, and put a fundamental steps for producing transgenic animals.1.We cultured the bovine ear fibroblast using DMEM with 10% FBS, then transfered the extra hTERT gene to cells of this type through the lipofactamine 2000. After 48h, we examined the expression of EGFP using fluorescent microscope and screened the clone using 600 ug/mL G418, then maked the clone propaganda using 300 ug/mL G418.2.Derived RNA from positive clone cells and designed the particular primer to go to the RT-PCR step. At last, the positive cells show the aim band (443bp), the negative cells have no the same band. The activity of telomerase of TN12 and TN22 is positive and that of the non-transfected cells is negative.3.Compared with the growth curve and the population doublings index of TN12, TN22, N12, N22. The population doublings index of TN22 (3.27±0.09) and N12 (3.14±0.06) have insignificant difference (p>0.05); TN12 (4.14±0.13) and N22 (2.06±0.05) compared respectively with three others have significant difference (p<0.05). The results showed that the lifespan of transfected cells are longer than that of non-transfected cells. However, the positive cells just transformed the senescence period (about 50PDs), cannot transform the crisis period (about 80PDs), so they cannot become immortalization. Through the examination of Flow cytometry, the rate of apoptosis between positive (0.111%) and negative cells (2.083%) have significant difference, and have normal karyotype.4.Using 21PDs negative cells ( N7) and 55PDs positive cells(TN14) which reached 100% confluence as donor cells for nuclear transfer, and compared the rate of cleavage, blastocyst and blastomere numbers. The results showed that the rate of cleavage and blastocyst between N7 and TN14 cells respectively have no significant difference, but the blastomere numbers of TN14 are lower than that of N7. This experiment demonstrated that bovine ear fibroblast transfected with hTERT gene can extend the lifespan and can be used as the donor cells for nuclear transfer.