【作者】 刘飞；

【导师】 范立强；

【作者基本信息】 华东理工大学 ， 生物化学与分子生物学， 2015， 硕士

【摘要】 基因打靶和RNA干扰是基因功能研究中的两种重要手段,为了在特定的组织和特定的发育时期内精确研究基因功能,通常将组织特异性或诱导性启动子与靶基因相融合,在特定的组织细胞中引导基因的敲除或沉默。肾脏雄激素调控蛋白(KAP)基因是一种在肾小管上皮细胞中特异性表达的基因,其表达受到雄激素、雌激素和垂体激素等多种激素的调控。本实验室通过截短体分析获得了 147 bp的最小启动子片段Kap147,并在人血管紧张素(hAGT)基因中鉴定了一个1.4kb的增强子片段(hAGT-Kpn),体外细胞转染实验证明Kap147/Kpn杂合启动子在不改变细胞特异性的前提下具有比Kap147启动子更强的转录活性和更敏感的雄激素调控性。在此基础上,本论文将Kap147/Kpn杂合启动子与Cre重组酶基因相融合构建转基因小鼠,在活体水平上研究Kap147/Kpn杂合启动子的表达特性。通过转基因鼠的繁殖鉴定,成功培育了 Cre-FO-1、Cre-F0-2和Cre-F0-3三个独立的转基因小鼠系;经RT-PCR、荧光定量PCR及免疫组化分析证实在Cre-F0-2小鼠睾丸组织中特异性地存在Cre基因的表达,在其他转基因小鼠系及其他组织中均未发现表达;免疫组化实验证明Kap147/Kpn杂合启动子将Cre基因的表达特异性地定位于睾丸靠近附睾组织处的生精小管中;通过与RFP/EGFP荧光报告鼠杂交证明Kap147/Kpn杂合启动子引导的Cre重组酶可以有效在小鼠睾丸组织中进行基因重组,是一种可用的睾丸组织特异性基因打靶工具鼠。荧光定量PCR分析了 Cre基因在不同年龄小鼠睾丸中的表达变化。结果表明,Cre基因的表达随年龄的变化呈现出成年鼠表达量高,幼年和老年鼠表达量低的现象,暗示Cre基因的表达可能受到性激素的调控;比较Cre基因和内源性Kap基因在不同小鼠间的表达情况,证实Cre基因与内源性Kap基因的表达存在一定的相似性;直接对小鼠注射雄激素使Cre基因的表达上升约1.5-2倍,而注射雄激素抑制剂则使Cre基因的表达降低30%-70%,进一步证实Kap147/Kpn杂合启动子在小鼠体内仍然受到雄激素的调控。成功构建了Kap147/Kpn杂合启动子引导绿色荧光蛋白shRNA干扰序列表达的重组质粒,细胞实验初步探索了Kap147/Kpn杂合启动子在RNA干扰中的效果。荧光观察和RT-qPCR检测证实Kap147/Kpn启动子引导的干扰序列特异性地在LLC-MK2细胞中使egfp基因的表达降低约40%,而在其他细胞中无明显干扰效果,表明Kap147/Kpn杂合启动子在细胞特异性地引导RNA干扰方面有一定的应用潜力。更多还原

【Abstract】 Gene targeting and RNA interference are two useful tools for gene function analysis.In order to precisely study gene function in specific tissues and specific developmental period,tissue specific or inducible promoter guided gene knockout or silence are often used.Expression of kidney androgen regulated protein(KAP)is specific in kidney proximal tubule cell-specific and androgen-regulated in vivo.In previous studies,the minimal Kap promoter(Kap147)and a 1.4 kb hetergenerous enhancer to Kap promoter,named hAGT-Kpn,between exons 2 and 3 of human angiotensinogen(hAGT)gene were identified by gene trunction and in vitro transfection experiment.In order to verify the function of Kap147/Kpn chimeric promoter in vivo,in this thesis,transgenic mice with ere recombinase(Cre)as the reporter gene were constructed,their expression characteristics were studied.Also,the posibility of using Kap147/Kpn chimeric promoter to direct cell-specific RNA interference was tested.Three independent transgenic mice,Cre-F0-1,Cre-F0-2 and Cre-F0-3,were obtained.Unfortunately,none of them showed kidney-specific Cre expression.Instead,we found tissue-specific Cre gene transcription and translation in the testis of the offspring of Cre-F0-2 by reverse transcription PCR,fluorescence quantitative PCR and immunohistochemistry.Immunohistochemistry also showed that Cre expression was targeted in the seminiferous tubules near epididymis.After crossing the offspring of Cre-F0-2 with a RFP/EGFP Cre reporter mice,we observed testis-specific gene recombination in their offsprings,which indicated that the offsprings of Cre-F0-2 are useful testis-specific gene targeting transgenic tool mice.Kap147/Kpn chimeric promoter directed Cre expression in the testis of transgenic mice Cre-FO-2 was regulated by androgen.Fluorescence quantitative PCR assay showed that the amount of Cre mRNA in adult mice was 20-30 times higher than that in newborn or senile mice.The transcription of Cre gene was similar to that of endogenerous Kap gene.Compared to uninjected normal mice,injection testosterone to female and male mice resulted in 1.5-2 fold increasment of Cre mRNA,while injection cyproterone acetate resulted in 30%-70%decreasment of Cre mRNA.Kap147/Kpn chimeric promoter can be used for kidney proximal tubule cell-specific RNA interference.Recombinant green fluorescent protein(EGFP)siRNA plasmids harboring Kap147/Kpn chimeric promoter and three different transcription stop signals were successfully constructed.Cell transfection experiments showed that the transcription and expression of EGFP was only reduced about 40%in LLC-MK2 cells(kidney proximal tubule derived epithelial cells),but not in Cos-7 cells(kidney derived fibroblast-like cells)and HeLa cells(cervical cancer derived epithelial cell).更多还原