A simple PCR-based method for the rapid genotyping of inherited fifth complement component (C5)-deficient mice.
A simple PCR-based method for the rapid genotyping of inherited fifth complement component (C5)-deficient mice.
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一种基于 PCR 的简单方法，用于遗传性第五补体成分 (C5) 缺陷小鼠的快速基因分型
DOI：
10.1538/expanim.14-0085
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发表时间：
2015
影响因子：
2.4
通讯作者：
Hu W
中科院分区：
文献类型：
--
作者：
Wang Q;Wang N;Zhang X;Hu W
补体的第五组分（C5）被认为是补体活化和功能的中心。然而，目前还没有基因工程敲除该基因的小鼠，而且仅有的市售遗传性c5缺陷小鼠，其编码框架中存在“TA”核苷酸缺失，是C57BL/10Sn遗传背景，而不是通常使用的C57BL/6和BALB/c。因此，这些老鼠必须回交到所期望的遗传背景。在这里，我们开发了一种ARMS（扩增难突变系统）PCR方法，使用特定的引物对，当所得产物通过琼脂糖凝胶电泳分析时，能够区分基因型。这些结果得到了定量RT-PCR和半定量PCR的支持，并与各回交代测序结果一致。利用ARMS-PCR方法，我们在9代回交中产生了C57BL/6背景下的c5缺陷小鼠，并使用补体介导的溶血试验进一步验证了表型。在这项研究中，我们描述了一种简单、快速和可靠的基于pcr的方法对遗传性c5缺陷小鼠进行基因分型，该方法可用于将C57BL/10Sn小鼠回交到其他遗传背景中。
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.