【作者】 周智慧；

【导师】 岑宁； 高波；

【作者基本信息】 扬州大学 ， 动物营养与饲料科学， 2010， 硕士

【摘要】 为了构建“睡美人”转座子(Sleeping Beauty transposon,SB)整合型鸡输卵管特异表达载体,根据已经发表的鸡卵清蛋白基因5’-和3’-调控区序列设计两对引物,用高保真PCR法从白来航鸡基因组DNA中分别扩增出长度各为3.0kb的卵清蛋白基因5’-和3’-调控区,将扩增产物克隆入pMD19-T载体,部分序列测定结果证明与国外发表的鸡卵清蛋白基因相应区域同源性为100%。将鸡卵清蛋白基因5’-和3’-调控区克隆入切除CMV启动子和SV40序列的pcDNA3.0载体,获得的鸡输卵管特异表达载体命名为pOV。分别将绿色荧光蛋白(EGFP)报告基因和人乳铁蛋白(hLF)cDNA通过MIU I位点克隆入pOV载体,获得重组载体pOV-EGFP和pOV-LF,然后用限制性内切酶将OV-EGFP和OV-LF表达盒从上述两个载体中切出,克隆至SB转座子载体pT2/HB的多克隆位点,从而构建成表达EGFP和hLF的整合型鸡输卵管特异表达载体pT2/HB-OV-EGFP和pT2/HB-OV-LF。为了证明构建的SB整合型载体能有效驱动目的基因在鸡输卵管上皮细胞中整合表达,将pT2/HB-OV-EGFP与SB转座酶按比例混和,经多聚阳离子(PEI)包裹后,注射至产蛋鸡输卵管膨大部,然后将载体注射鸡扑杀,在不同时间段取输卵管注射部位,分别用RT-PCR和组织冰冻切片检测报告基因的表达,结果显示EGFP基因在输卵管注射部位获得有效表达,且注射后45d表达水平仍较高。提取输卵管注射部位基因组DNA,用PCR和点杂交检测报告基因的整合,结果显示LF基因获得有效转座整合。为了检测hLF cDNA的表达性能,用上述方法将pT2/HB-OV-LF注射至产蛋鸡输卵管膨大部,在不同时间段取输卵管注射部位,RT-PCR和免疫组织化学检测LF结果表明,hLF基因在输卵管膨大部获得正确转录和翻译,表达产物能够与hLF单克隆抗体发生抗原-抗体反应。这些试验结果表明,本研究构建的整合型输卵管特异表达载体不仅能有效驱动目的基因在产蛋鸡输卵管上皮细胞中表达,而且能够促进转基因与受体染色体的整合,可以用于转基因鸡输卵管生物反应器的研制。为了进一步研究SB转座子能否有效介导目的基因在鸡精原干细胞中发生转座整合,本研究又构建了SB整合型组织特异表达载体,即用限制性内切酶AseⅠ和AflⅡ将CMV-EGFP表达盒从质粒pEGFP-N1中切出,克隆至SB转座子载体pT2/HB的多克隆位点,构建成表达EGFP报告基因的整合载体pT2/HB-CMV-EGFP。然后将pT2/HB-OV-EGFP与SB转座酶按比例混和,经多聚阳离子(PEI)包裹后,通过睾丸网注射至成年公鸡曲精细管内,然后在不同时间段取睾丸注射部位,分别用冰冻切片、免疫组织化学和RT-PCR等方法检测报告基因的表达。结果显示EGFP基因在曲精细管获得稳定表达,注射后40d仍然具有较高表达水平。提取睾丸注射部位基因组DNA,分别用PCR和点杂交检测报告基因的整合,结果显示EGFP基因在注射后不同阶段均可获得转座整合。这些试验结果表明,本研究将SB整合技术和睾丸网注射法进行睾丸内精原干细胞转染技术相结合,可以实现外源基因的有效整合和稳定表达,为建立SB转座子介导法制作转基因鸡奠定了坚实的基础。更多还原

【Abstract】 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.更多还原