Effect Of Rapamycin On The Expression Of Exogenous Gene In Transgenic Cells Of Cashmere Goat

Rapamycin, which is also known as Sirolimus, belongs to the macrolide antibiotic family. Rapamycin was separated from the metabolites of Streptomyces hygroscopicus AY B-994, and AY B-994 was got from an soil sample of the Easter Island in the South Pacific Ocean. Rapamycin was initially used as antifungal drugs. But finally rapamycin has been used for the treatment of organ transplant rejection, because it was found that it had a strong inhibitory effect on immunity. In mammals, mTOR forms two distinct complexes, mTORC1 and mTORC2, which differ in their composition and sensitivity to rapamycin. Rapamycin is a specific inhibitor of mTORC1. Compared with mTORC1, mTORC2 is insensitive to rapamycin. mTORC1 regulate protein synthesis controlled through downstream target protein 4E-BP1 and S6K, and also is involved in the regulation of transcription. Although the function of mTOR pathway has a more profound study, but the role of mTOR pathway on exogenous genes in transgenic cells has not been reported.In the present study, the human keratin 5 promoter was firstly cloned and the function of it was examined in the cashmere goat cells with pK5-DsRed2-1 vector. Secondly, the skin specificity expression vector, pCDsRed-KI was constructed by subcloning IGF-1 gene into pCDsRed-K5. The transgenic cells were got by using pCDsRed-KI to transfect the cells. Finally, transgenic cells was treated with rapamycin and the expression of exogenous gene was examined with RT-PCR.The results show that:1) the human keratin 5 promoter has the skin specificity in the cells of cashmere goats; 2) IGF-I gene can be expressed in the skin fibroblast cells, and the transgenic cell line was got through screening the skin fibroblast cells; 3) rapamycin can inhibite the transcription of exogenous gene IGF-1. The result shows that mTOR pathway might have regulation effect on the expression of the exogenous gene in transgenic cells.