A simple PCR-based method for the rapid genotyping of inherited fifth complement component (C5)-deficient mice

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  • WANG Qingkai
    Fudan University Shanghai Cancer Center and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, P.R.China
  • WANG Na
    Fudan University Shanghai Cancer Center and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, P.R.China
  • ZHANG Xin
    Fudan University Shanghai Cancer Center and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, P.R.China
  • HU Weiguo
    Fudan University Shanghai Cancer Center and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, P.R.China Department of Immunology, Shanghai Medical College, Fudan University, Shanghai 200032, P.R.China

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抄録

The fifth component of complement (C5) is considered to be the center of complement activation and function. However, there are no genetically engineered knockout mice for this gene, and the only commercially available inherited C5-deficient mice, in which a “TA” nucleotide deletion in the coding frame was previously identified, are in theC57BL/10Sn genetic background rather than the commonly used backgrounds C57BL/6 and BALB/c. Therefore, these mice must be backcrossed into the desired genetic background. Here, we developed an ARMS (amplification refractory mutation system) PCR method using a specific primer pair that was able to discriminate between the genotypes when the resulting product was analyzed by agarose gel electrophoresis. These results were supported by quantitative RT-PCR and semi-quantitative PCR and were consistent with the results from sequencing each backcrossed generation. Using ARMS-PCR method, we generated C5-deficient mice in the C57BL/6 background over 9 backcrossed generations and further verified the phenotype using complement-mediated hemolytic assays. In this study, we describe a simple, rapid and reliable PCR-based method for genotyping inherited C5-deficient mice that may be used to backcross C57BL/10Sn mice into other genetic backgrounds.

収録刊行物

  • Experimental Animals

    Experimental Animals 64 (3), 261-268, 2015

    公益社団法人 日本実験動物学会

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