Construction Of Chicken RNAi Avian Retroviral Vector And Its Application In Functional Study Of Gene BAF57

The mammalian SWI/SNF chromatin remodeling complex, which plays importance roles in transcriptional regulation, contains approximately 10 protein components including BRG1, BAF155, BAF170, BAF60 and BAF57. It was shown that BAF57 can interact directly with estrogen receptor alpha (ER-α) and the androgen receptor (AR) and help recruit the SWI/SNF complex to estrogen-and androgen-responsive promoters for hormone-dependent transcriptional activation. We also found BAF57 were differentially expressed in the early male and female embryonic development. This study constructed RNAi vector using avian retroviral vector RCASBP integrated with U6 promoter and RNA interfereed gene BAF to explore its role in gonadal development and its network in the process of regulation. Results are as follows:1. By using one step PCR method chicken U6 promoter (CU6-4) was obtained, the shRNA from 9 to 11 day old White Leghorn chicken genomic DNA and GFP from plasmid pBI-EGFP was sequently linked with retrovirus RCASBP——a complete RNAi vector was constructed. After transfection of viral DNA, viral packaging, collection and extraction, ultimately, we get the virus to infection. The results showed that the viral vector was constructed successfully, and show one Step PCR method is simple, effective way.2. The virus obtained by packaging were carried out to infect cells and chicken embryos for observing the infection. The results showed that cells infected with good effection, expessed strong fluorescence. After 48-72h transfection, Fluorescence began to express, and after culturing 5 to 7 days cells are collected, extracted RNA, and used to do semi-quantitative measurement. Before Chicken are infected, use adhesive tape cut method to open the window. After the injection of virus, culturing 5 to 7 days to observe the chicken can see strong fluorescent expression, but chicken embryos infected all died.3. Infected cells are collected, extracted RNA, reverse transcription, semi-quantitative detection of the expression of BAF57 and Sex differentiation associated gene-Sox9, Dmrt1, AMH, Aromatase, AR and ER-a. Results show, the expression level of target gene BAF57 decline, the expression of ER-a (estrogen receptor) and AR (androgen receptor) are reduced, and Sox9 expression also appeared reduced, but We did not detect To Dmrt1, AMH and aromatase gene expression (DF-1 cells did not have)The research successfully completed preliminary study of chicken Candidate genesBAF57 in gonadal development using of the retroviral vector, and provided basis for further study. Meanwhile, by bring the retroviral vector into our lab, the experiment improve the laboratory RNAi technologiy platform.