TauT转基因大鼠的构建及鉴定

Establishment and identification of TauT transgenic rats

目的:为研究牛磺酸转运体(TauT)在神经系统疾病中的作用,构建并繁育脑特异性表达TauT转基因模型大鼠(TauT+/-大鼠)。方法:将TAUT cDNA插入脑特异性PDGF启动子下游,构建转基因表达载体,显微注射法建立TAUT转基因大鼠。Genotype鉴定转基因大鼠的基因型。经过配种繁殖和鉴定后,绘制2~8月龄生长曲线、测定各脏器质量、脏体系数和脏脑系数;RT-PCR技术检测TauT+/-大鼠大脑组织中Slc6a6 mRNA基因表达水平;Western blot检测TauT+/-大鼠在大脑、心、肝组织的TauT蛋白表达,并对TauT+/-大鼠大脑组织进行免疫组化染色和HE染色,IPP软件对免疫组化图像进行处理。结果:成功构建PDGF-Slc6a6表达质粒,得到3只TauT+/-大鼠F0代。经过繁殖和鉴定,与同窝阴性大鼠(TauT-/-大鼠)比较,TauT+/-大鼠大脑组织Slc6a6 mRNA基因表达水平显著升高,大脑、心、肝组织TauT蛋白表达水平没有显著性差异,HE染色和免疫组化结果显示TauT+/-大鼠脑组织海马区和皮层区未见异常,TauT蛋白表达没有显著性差异。结论:成功构建TauT转基因大鼠品系,为后续研究TauT在大脑中的作用提供一种较好的动物模型。

Objective:To establish brain-specific expression taurine transporter transgenic rats(TauT+/-rats),which can be used for the study of the effects of taurine transporter(TauT)on neurological diseases.Methods:The transgene expression vector was constructed by inserting the TauT cDNA into the downstream of PDGF promoter specifically expressed in brain tissue.The transgenic rats was established by micro injection and detected the genotype by PCR.The weight curves of 2-8 months TauT+/-ratswere measured,the weight of each organ,the visceral coefficient and the visceral brain coefficient were measured.The expression of Slc6a6 mRNA in the brain tissue of TauT+/-rats was detected by RT-PCR.Western Blotting was used to detect TauT protein expression in brain,heart and liver tissues.Immunohistochemical staining and HE staining were performed on TauT+/-rat brain cortex and hippocampus,and IPP processing Immunohistochemical images.Results:TauT cDNA transgenic vector was successfully constructed and three TauT transgenic rats were established.The expression level of Slc6a6 mRNA in brain tissue of TauT+/-rat was significantly increased,but the expression levels of TauT protein in brain,heart and liver tissues were not.HE staining and immunohistochemistry showed no abnormalities and significant difference in brain cortex and hippocampus.Conclusion:The brain-specific expression TauT transgenic rat was successfully established and identified.The transgenic rat could be a useful model to research the function of taurine transporter in brain.