Production Of Bovine Fibroblast Cells For Insulin-Like Growth Factor-Ⅰ Overexpressed And Knocked-Down

It is reported that Insulin-like Growth Factor-I (IGF-I) plays a key role in mammalian embryonic development. Appropriate concentration of IGF-I raises oocytes maturation rate in vitro. Due to the anti-apoptosis effects, IGF-I advances embryo survival and increases the inner mass's number of blastocyst. However the molecular mechanism has not been clarified. As two important methods for the functional genomics research, transgenetic animals and RNA interference have been used to study the mechanism of genes expressed in the process of embryonic development. In order to indicate the molecular roles of IGF-I during the bovine preimplantation embryonic development in vitro, an overexpression vector, pIRES2-EGFP-IGF1 and an interference vector, pGCsi-IGF1 were introduced to the bovine fibroblast cells (BFFCs) by using lipofectamine and electroporation, then the transgentic BFFCs were screened by the G418 and Hygromycin B to establish the cell lines of IGF-I steady expressed. The cell lines could be the nuclear donor to produce transgenetic embryo models for IGF-I overexpressed and kncked-down, moreover this might make a great effort for the advanced study.1. The construction of the overexpressin vector pIRES2-EGFP-IGF1 and the interference vector pGCsi-IGF1.(1) The IGF-I coden sequence was obtained from bovine total RNA by RT-PCR, then linked with T vector pMD19-T to get an intermediate construction pMD19-T-IGF1. At next step, the overexpression vector pIRES2-EGFP-IGF1 was accomplished by linked IGF-I fragment on the double cistron vector pIRES2-EGFP between BglII and SalI sites. The structure and sequence are consistent with expectation based on restriction endonucleasa assay and sequencing results.(2) Hairpin RNA interference fragment was designed, based on 594-622bp 5'-GGCTCAGAAGGAAGTACAT-3' area of IGF-I gene, then linked with pGCsilencer-RFP vector to accomplish the interference vector pGCsi-IGF1.2. Isoation and In vitro culture of bovine fetal fibroblast cellsThe bovine fetal fibroblast cells (BFFCs) were successfully isolated by attachment of tissue pieces from a bovine fetus. The isolated cells were purified and proliferated in DMEM+10%FBS at 37℃in a humidified 5% CO₂ incubator. Then the cells were cryopreserved within DMEM+20%FBS media supplemented 10% of DMSO. Morphology and growth character were observed and recorded, the growth curves of the cells within 5^st and 10^st passages were drawed and chromosomal analysis of the cells with 5^st passage were performed. The results showed that the growth speed was slow down with the passage number increased, and 5^st passage cells contained normal chromosome number consisting of 60 chromosomes (2n=60) and can be used for tansfection. Meanwhile, toleration of the BFFCs on G418 and Hygromycin B were examined respectively, the optimal screening concentration of G418 and Hygromycin B were 800μg/ml and 50μg/ml.3. Gene transfection into bovine fetal fibroblast cells and identity of transgenetic clonesThe BFFCs were transfected by lipofectamine and electropored respectively. After 48 hours, the effects of vectors were analyzed by Real-Time PCR. The result indicated that, compared with the negative control, the expression of IGF-I in the BFFCs transfected by pIRES2-EGFP-IGF1 significantly increased (40 folds, P<0.05); the BFFCs transfected by pGCsi-IGF1 expressed lowest IGF-I mRNA (0.023 folds, P<0.05). These results confirmed that both the vectors had rationality and validity as well as the biological function. Then the BFFCs transfected by the two vectors were screened by 800μg/ml G418 for pIRES2-EGFP-IGF1 and 50μg/ml Hygromycin B for pGCsi-IGF1. After selected for 10-12 days at 37℃in a humidified 5% CO₂ condition, clones with green fluorescent and red fluorescent were obtained respectively. Identification of the genomic DNA of transgenetic cells by PCR proved that the exogenous gene have integrated into geneomes of the cells.