The Pre-Exploration Of Establishing Chicken Oviduct Bio-Reactor By Retrovirus

Because of it's special advantages the research of chicken oviduct bioreactor has been one of the foci of transgenic studies and pharmacy.At first, in this research, we explored the primary culture of the chicken epithelial cell of 17 months, 6-9 months chicken and the hormone stimulated young chicken. We try to find out which method infected the oviduct epithelial cell effectively. Then we had tried Lipofectin infected method, CaCl2 infected method and the electro-pulse infected method and found the last method is the best.We regard the eGFP as report gene and the hEPO as the subject gene in this research. We PCR amplified the 1.1kb chicken ovalbumin 5'-flanking regulatory region (OV), GFP, SGFP and EPO DNA sequence and cloned them into pMD-18T vector. The right ligation had been sifted. Then through digestion and ligation we observe the right ligation of OV with GFP, sGFP and EPO. At last the right ligated DNA sequence had been digested and ligated into retrovirus vector pLNCX. We observe the right retrovirus vector of pLN-OV-GFP, pLN-OV-sGFP and pLN-OV-EPO.The end, the vector expression MLV's Gag and Pol gene infected the HEK 293 cell. We gained the right cell clones through sifted by HisD (L-Histidinol dihydrochloride). Then we extracted the genome of the cell and PCR detected it. Then we infected the cell with retrovirus expression vector pLN-OV-GFP,or pLN-OV-sGFP. After sifted by G418 we extracted the cell's genome and PCR detected. We gained the retrovirus packaging cell line that integrated with report gene.In summery, we had explored the primary culture of the chicken epithelial cell of different chicken ages and had detected different infected methods. Three retrovirus vector had been constructed successfully and the retrovirus packaging cell line HEK 293 that can express MLV's Gag and Pol gene and pLN-OV-GFP or pLN-OV-sGFP has gained. We do these in order to detect the chicken oviduct bioreactor system's construction and establish the base for chicken oviduct bioreactor that specific express hEPO.