荧光定量RT-PCR技术分析人地中海贫血β^(654)珠蛋白基因转基因小鼠mRNA的表达

Analysis of mRNA Expression in Human β^(654) Globin Gene Transgenic Mice by Fluorescent Quantitive RT-PCR

目的采用荧光定量RT-PCR技术检测所制作的人地中海贫血茁654珠蛋白基因转基因小鼠模型mRNA的表达情况,评价该技术应用于小鼠mRNA分析的价值。方法根据GenBank小鼠mRNA序列,设计用于扩增小鼠mRNA的引物和反向探针,构建荧光定量RT-PCR技术。实验分两组,分别对转基因小鼠和正常小鼠中mRNA进行检测。采用SPSS统计软件,分析和比较两组动物中mRNA的表达量及表达的稳定性。结果所检测的11只转基因阳性小鼠中检测出人茁654珠蛋白基因mRNA和小鼠内源性mRNA;而在33只正常小鼠中只检测到小鼠内源性mRNA。荧光定量RT-PCR技术可检测到105拷贝数的人茁654珠蛋白基因mRNA和115拷贝数的小鼠内源性mRNA。荧光定量RT-PCR技术分别在F1代和F2转基因阳性小鼠中稳定地检测到,表明转基因小鼠mRNA获得稳定表达。结论所构建的荧光定量RT-PCR技术分析转基因小鼠mRNA表达具有检测定量、敏感、高效快捷的特点,该方法在鉴定和评估转基因小鼠mRNA的表达中稳定性好、定量准确,具有非常重要的应用价值。

Objective To establish fluorescent quantitative reverse transcription polymerase chain reaction （RT-PCR） to detect mRNA expression in human β^654 globin gene transgenic mice and evaluate its application value on analyzing mRNA expression of transgenie mice. Methods The primers and reverse probe were designed according to the sequences published in GenBank. A fluorescent quantitative RT-PCR method was developed. Blood samples were collected from 11 human β^654 globin gene transgenic mice and 33 normal mice . The expression levels of human β^654 globin gene mRNA and the mouse endogenous mRNA were investigated by fluorescent quantity RT-PCR. PCR products were cloned into PUC19 vector and sequenced. The copy numbers of human 13~ globing gene mRNA and the mouse endogenous mRNA were used to estimate the expression level and the sensitivity of the method. Results Both human 13~4 globin gene mRNA and the mouse endogenous mRNA were detected in all the 11 transgenic mice, which had confirmed by polymerase chain reaction （PCR） and Reverse dot blot hybridization （RBD）. In all the 33 normal mice, only the mouse endogenous mRNA was detected but not the human β^654 globin gene mRNA. 105 clones of human β^654 globin cDNA and 115 copies of the mouse endogenous cDNA were detected in recombinant plasmids. The human β^654- globin gene was stably expressed in the transgenic mice in two generations, including the founders and the secondlygeneration. Conclusion The fluorescent quantitative RT-PCR method was rapid, sensitive, highly specific and very useful to estimate the expression level of the transgene in transgenic mice.