Title: Analysis of mRNA Expression in Human β~(654) Globin Gene Transgenic Mice by Fluorescent Quantitive RT-PCR
Abstract: Objective To establish fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR) to detect mRNA expression in human β654 globin gene transgenic mice and evaluate its application value on analyzing mRNA expression of transgenic 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 β654 globin gene transgenic mice and 33 normal mice . The expression levels of human β654 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 β654 globing gene mRNA and the mouse endogenous mRNA were used to estimate the expression level and the sensitivity of the method. Results Both human β654 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 β654 globin gene mRNA . 105 clones of human β654 globin cDNA and 115 copies of the mouse endogenous cDNA were detected in recombinant plasmids. The human β654-globin gene was stably expressed in the transgenic mice in two generations, including the founders and the secondly generation. 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.
Publication Year: 2005
Publication Date: 2005-01-01
Language: en
Type: article
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