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目的构建载体实现miR-29c在肾脏组织的特异性表达。方法分别以质粒pc DNA 3.1(+)和小鼠基因组DNA为模板,扩增CMV增强子序列和肾脏特异性钙黏着素蛋白KSP-cadherin启动子序列;将连接成的单一片段酶切后替换p CAGGS载体上的CAG启动子构成目的载体p CMV-KSP。将GFP编码区序列或小鼠miR-29c前体及两侧序列克隆到构建的载体上观察转染后目的基因的表达。结果从相应的模板上扩增出的序列经验证后连接到了p CAGGS载体上,转染细胞后在荧光显微镜下观察到KSP启动子调控下的GFP在MDCK中表达,而在NIH-3T3细胞中仅见微弱荧光。p CMV-KSP-p CAGGS-miR-29c载体较对照载体在转染人胚肾293T细胞24 h后,成熟miR-29c的表达显著升高,其靶基因bcl-2表达相应降低。结论构建KSP启动子调控miR-29c表达的载体,为构建肾脏特异性表达miR-29c转基因小鼠,进一步研究miR-29c在肾移植后的各种病理过程中的作用奠定了基础。
Objective To construct a vector for specific expression of miR-29c in renal tissues. Methods The promoter region of CMV enhancer and KSP-cadherin promoter were amplified by using pcDNA 3.1 (+) and mouse genomic DNA respectively. The single fragment ligated was replaced by p The CAG promoter on the CAGGS vector constitutes the destination vector pCMV-KSP. The GFP coding region sequence or the mouse miR-29c precursor and the sequences on both sides were cloned into the constructed vector to observe the expression of the target gene after transfection. Results The amplified sequences from the corresponding templates were verified to be connected to the pCAGGS vector. After transfection of the cells, GFP under the KSP promoter was observed under a fluorescence microscope in MDCK, whereas in NIH-3T3 cells Only see weak fluorescence. The expression of mature miR-29c in pCMV-KSP-p CAGGS-miR-29c vector was significantly higher than that in the control vector after transfected into human embryonic kidney 293T cells for 24 h, and its target gene bcl-2 expression decreased accordingly. CONCLUSION: The construction of KSP promoter to regulate the expression of miR-29c has laid the foundation for the further study of the role of miR-29c in various pathological processes after kidney transplantation in order to construct kidney-specific transgenic mice expressing miR-29c.