The Optimization Study Of Chicken Oviduct Specific Promoter

The study of transgenic animal was begun from1970s, and made great achievements, including development of transgenic technology and its applications. The research of transgenic animal bioreactors, especially mammary gland bioreactor has made much progress, which provides a novel way to produce medicine proteins. Poultry oviduct bioreactors were also been one foci of transgenic research in various countries, because of the outstanding egg-laying capacity and other advantages of chicken. However, the strategy to achieve high expression level of foreign gene in oviduct-specific manner was remained as the main technical bottleneck. We selected the chicken ovalbumin gene5’upstream regulatory region as a promoter candidate for future transgenesis of chicken bioreactors. In this study, various experiments for optimization of this promoter were carried out. Some aspects of this research are as following:1. The cloning of5’-flanking sequence of chicken ovalbumin geneSix different5’-flanking sequences (OV1-OV6) of chicken ovalbumin gene were successfully amplified by the method of PCR. The lengths of these sequences are959,1381,2081,3140,5474and6293bp, respectively. All fragments contained the ovalbumin gene core promoter.2. The construction of a series of promoter-reporter vectorThe eukaryotic expression vectors of pGL4-OVs were constructed by using pGL4.10plasmid as backbone. The result of restriction enzyme digestion and sequencing showed that six luciferase expression vectors under control of OV1-OV6has been constructed successfully.3. The establishment of primary chicken oviduct epithelial cell culture system We separated chicken oviduct epithelial cells from female chicks treated with estrogen and/or laying hens. In this period, the technical methods at some aspects of culture were explored to gain high purity oviduct epithelial cells, such as oviduct separation, trypsin digestion, pre-incubation and cell transfer. Thus, we provided an experimental platform to access tissue-specific expression of foreign genes in chicken oviduct epithelial cells.4. The expression regulative property of the ovalbumin promoter fragmentsIn order to test the gene expression activity and specificity under control of different ovalbumin promoter fragments, the primary oviduct epithelium cells, DF-1cells and chicken embryo fibroblasts cells were transfected with the six promoter-reporter plasmids. Luciferase activities of these six vectors were detected and compared to identify the optimal vector construction. The result showed that the pGL4-OV3plasmid possesses the strongest expression activity and better tissue-specificity among all vector constructs. This assay also reveals some potential sequence regions of enhancers, repressors and sequence-specific regulatory elements. The result shows:chicken oviduct-specific and repressor-like regions were implicated between-3102-2042,-1342-921and-5436-3102; chicken oviduct-specific and enhancer-like regions were implicated between-2042-1342and-6233-5436. And there is no chicken oviduct-specific region between-921-+38.By this study, we not only selected an optimal promoter material for generation of transgenic chicken bioreactors, but also provided material and knowledge base for further developing more excellent poultry oviduct-specific promoters.