Research On The Establishment Of Reversibly Immortalized Sheep Fibroblast Cell Line And Its Applications

Animal cells cultured in vitro have important applications in fundamental biology research, cell therapy, gene modification and transgenic animal cloning. Nevertheless, primary cells isolated from animal tissues have limited proliferative lifespan and it’s not conducive to serial cultivation, so and not conducive to their applications in gene modification and transgenic animal cloning. It has been confirmed that ectopic expression of human telomerase reverse transcriptase subunit (hTERT) in normal diploid cells can reconstitute telomerase activity and extend their lifespan. However, the constitutive expression of hTERT may lead to developmental failure of cloned embryos or other problems. Given these, the present paper tries to establish reversible immortalized sheep fibroblast cells by conditional expression of hTERT, used for somatic cells transgenic animal cloning in livestock and other researches in future.Inducible hTERT expression plasmid vector pTet-on hTERT was constructed based on Tet-on system. Then, linearized pTet-on hTERT plasmids were transfected into sheep fetal fibroblasts and six single-cell clones with stable expression of hTERT were achieved eventually, cultured in medium supplemented with Dox. When expand to 60 mm dishes from 96 plates, each of the six cell clones was divided into two groups:one group cultured in Dox-supplemented medium (referred to as group h(+)), and the other group cultured in non-Dox medium (referred to as group h(-)). Expression levels of hTERT in h(+) and h(-) cells were analyzed by RT-PCR and Western blot, and it displayed that there were high levels expression of hTERT mRNA and hTERT protein in h(+) cells. Telomerase activity in h(+) and h(-) cells were detected by TRAP, and it showed that telomerase activity could be reconstructed in h(+) cells. After 120 days of serial passages, the population growth rates of h(-) cells gradually slowed and the cells entered senescence eventually, differently, the population growth rates of h(+) cells remained stable and the cells were in good shape. These results revealed that ectopic expression of hTERT can greatly extend the proliferative lifespan of sheep fetal fibroblasts. Subsequently, each group h(+) cells were subdivided into two groups:one group remained in Dox-supplemented medium (referred to as group h(+), still) and the other group cultured in non-Dox medium (referred to as group h(+-)). After another 130 days of serial passages, the population growth rates of h(+) cells were unchanged and the cells were vigorous, by contrast, the population growth rates of h(+-) cells gradually slowed and the cells were flat. It demonstrated that the immortalized cells can be reverted to a normal state. Soft agar assay and karyotype analysis were performed and the six group h(+) cell lines showed no transformed phenotypes after 250 days of serial passages.Furthermore, myogenic differentiation 1 (MyoD) was transferred into h2(+) cells, which had been cultured continuously for 200 days. After another round of cell clone selection,13 cell clones integrated with MyoD were achieved. Then, the expression of MyoD and some other muscle-specific genes were detected by RT-PCR. It suggested that the life extended cells could suffer another round of transgenic operation and so they could be applied to gene modification and fundamental cytological studies.In materials aspect, adult fibroblasts are more available and not involving in ethics issues. What’s more, as nuclear donor, adult fibroblasts can be used for the cloning of individual animals with advantageous traits as well as the propagation and breed conservation of the rare and endangered animals. Nonetheless, compared with fetal fibroblasts, the proliferation capability of adult fibroblasts is even worse and the efficiency of nuclear transfer is even lower. To address the problem, pTet-on hTERT plasmids were transfected into adult fibroblasts and a life extended cell line A3h38(+) was selected. Cell colony forming experiments were performed and it revealed that the single cell colony formation ability and colony amplification capability of A3h38(+) cells been cultured continuously for 136 days were stronger than primary adult fibroblasts. And somatic cell nuclear transfer (SCNT) were conducted and it indicated that the cloned blastocyst rate of A3h38(+) cells been cultured continuously for 80 days increased significantly compared with primary adult fibroblasts. Therefore, the establishment of immortalized fibroblasts will provide valuable materials for gene modification and transgenic cloning in livestock somatic cells.