Expression Of Human IGF Ⅰ Gene In PK-15 Cells

Insulin-like growth factorâ… (IGFâ… ) is not only a important regulatory factor for growth, development and etabolism in body, but also the major mediator for growth hormone(GH) activating growth. It has insulin-like and growth-like effect, promoting the growth of muscle cells differentiation, balancing of fat deposition. Research has shown that, human IGFâ… gene transfer pigs have mang strong points such as low fat content, high lean without the weak points in GH gene transfer pigs. Therefore, using advanced recombinant DNA-somatic cell nuclear transfer technology transfer hIGFâ… gene in pigs is important to study.This research references the published hIGFâ… gene sequence in GENBANK, using RT-PCR cloned hIGFâ… mature peptide gene sequence. Constructing pcDNA3.1-hIGFâ… eukaryotic expression vector within pCMV promoter, the neo and amp resistance gene, which may stably expressing the gene products in mammalian cells. By liposome plasmid transfected them into pig kidney epithelial (PK-15) cells, and suffering the G418 concentration gradient selected transgenic cloned cell lines. Finally, detect and identify the expression of foreign genes in transfected cells lines by realtime-PCR and ELISA.The main results are as follows:1. Have exactly cloned the hIGFâ… gene sequence encoding the mature peptide, the size of 315bp, consistent with the published sequences.2. Have successfully built the pcDNA3.1-hIGFâ… eukaryotic expression vector.3. By the concentration gradient of G418 screened, have got 8 transgenic cell lines.4. And the transgenic cell lines expresses hIGFâ… gene at both transcription and expression levels.These results suggest that exogenous hIGFâ… gene in PK-15 cells with stable expression of transgenic experiments for the follow-up provides a theoretical basis.