睡美人转座子介导外源基因在鸡输卵管和睾丸整合表达特性研究动物营养与饲料科学专业论文.docxVIP

周智慧“睡美人”转座予介导外源基因在鸡输卵管和睾丸整合表达特性研究 周智慧“睡美人”转座予介导外源基因在鸡输卵管和睾丸整合表达特性研究 Studies on transgene Expression and Integration in Chicken Oviduct and Testis Using Sleeping Beauty Mediated Gene Transfer M．S．candidate：Zhou zhi-hui Supervisor：Associate Prof．Cen Ning Instructor Gao Bo Abstract To construct Sleeping Beauty DNA transposon-based chicken oviduct-specific expression vectors，the 5-and Y-regulatory regions of chicken ovalburnin gene were amplified from chicken genome DNA by hi曲fidelity PCR using primers designed according to previously published sequences．The 3．0kb PCR products were subcloned into pMD1 9-T vector and the correctness of the sequences was confirmed by sequence analysis．The Y-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)relDorter gene and human lactoferrin mLF)were inserted into the downstream of the 5-regulatory region， respectively,and the resulted vectors were named aS pOV-EGFP and pOVoLF．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 Wel-e called pT2／HB--OV-EGFP and pT2／HB—·OV-LF． To evaluate wether the Sleeping Beauty DNA transposon-bascd vector call drive expression and integration of gene of interest in hen oviduct epithelial cells，following mixing wim polyethyleneimine，the recombinant vector pT2／HB—OV-EGFP and plasmid DNA encoding SB transposase pCMVl 6 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 一4 一4 扬州大学硕士学位论文 PCR and dot blotting analysis．The results showed that EGFP gene could be transposed and integated