Studies On Transgene Expression And Integration In Chicken Oviduct And Testis Using Sleeping Beauty Mediated Gene Transfer

To construct Sleeping Beauty DNA transposon-based chicken oviduct-specific expression vectors, the 5'-and 3'-regulatory regions of chicken ovalbumin gene were amplified from chicken genome DNA by high fidelity PCR using primers designed according to previously published sequences. The 3.0kb PCR products were subcloned into pMD19-T vector and the correctness of the sequences was confirmed by sequence analysis. The 5'-and 3'-regulatory regions were then subcloned into a modified pcDNA3.0 vector as a Sal I/Not I fragment, resulting in oviduct-specific expression vector pOV. Then enhanced green fluorescence protein (EGFP) reporter gene and human lactoferrin (hLF) were inserted into the downstream of the 5′-regulatory region, respectively, and the resulted vectors were named as pOV-EGFP and pOV-LF. The EGFP and hLF gene expression boxes were excised from the pOV-EGFP and pOV-LF vectors by restriction enzyme digection and subcloned into SB vector pT2/HB and the resulted recombinant vectors were called pT2/HB-OV-EGFP and pT2/HB-OV-LF.To evaluate wether the Sleeping Beauty DNA transposon-based vector can drive expression and integration of gene of interest in hen oviduct epithelial cells, following mixing with polyethyleneimine, the recombinant vector pT2/HB-OV-EGFP and plasmid DNA encoding SB transposase pCMV16 were coinjected into egg-dropping hens'oviducts via surgery and the injected tissure were collected for frozen sections and RT-PCR analysis. The results showed that stable EGFP expression was observed up to 45 days in the oviduct. And genome DNA was extracted from the injected tissure for PCR and dot blotting analysis. The results showed that EGFP gene could be transposed and integated into chicken chromosomes. To evaluate the cloned hLF cDNA expression property, the vector pT2/HB-OV-LF was injected into egg-dropping hens'oviducts with the same way and the injected tissure were collected for RT-PCR and immunohistochemistry - frozen sections(IHC-FR) analysis, the results showed that LF gene can be expressed correctly and the products could react with an antibody specific for human lactoferrin. These results indicate that the constructed SB- based vector could not only drive exogenous gene expression in laying hen oviduct epithelial cells, but also facilitate the transgene transposition and integration.To evaluate wether SB can mediate transgenes transposition in chicken sperm stem cells(SSCs), another SB-based tissure expression vector was constructed. The CMV-EGFP expression box was excised from the pEGFP-N1 vector by restriction enzyme digection and subcloned into pT2/HB vector and resulted recombinant vector was named pT2/HB-CMV-EGFP. Then pT2/HB-CMV-EGFP was effectively delivered to cock convoluted seminiferous tubule with pCMV16 via rete testis injection and the injected tissure were collected for frozen sections, IHC-FR and RT-PCR analysis. The results showed that EGFP gene were expressed stably in convoluted seminiferous tubule, and the EGFP expression was still strong up to 40 days. The results of PCR and dot boltting of the injected tissure genome DNA showed that EGFP gene could be transferred into chicken testis genome. Those results suggest SB is an efficient tool for transgenesis and expression in chicken.In conclusion, a SB-based chicken oviduct-specific expression vector has been constructed and an efficient chicken rete testis injection protocol has been established, which can be used for further studies on transgenic chickens.