Genotyping Genetically Modified (GM) Mice.
Genotyping Genetically Modified (GM) Mice.
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对转基因 (GM) 小鼠进行基因分型。
DOI：
10.1007/978-1-4939-9837-1_12
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发表时间：
2020
期刊：
影响因子：
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通讯作者：
Parker-Thornburg,Jan
中科院分区：
文献类型：
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作者：
Aryal,NeerajK;Parker-Thornburg,Jan
在生成新的小鼠模型之前，重要的是计划用于检测所生成的哪些小鼠具有所需突变的方法。几乎所有类型的突变都可以使用 PCR 进行检测。然而，引物的选择将根据用于生成模型的方法而有所不同。转基因小鼠应通过独特的连接片段进行基因分型。用于产生敲除或敲入小鼠的靶向 ES 细胞应使用来自构建体中独特标记和构建体外部区域的引物进行基因分型。 ES 细胞中的靶向也可以使用基因组 Southern 印迹来检测。使用 CRISPR/Cas9 靶向的小鼠应使用 PCR 扩增感兴趣的区域，然后通过 Surveyor Assay（针对基因敲除和点突变）评估大小变化（针对序列的较大变化）和/或进行测序以验证突变。这些模型中的每一个对基因分型都有独特的要求，如果不了解这些要求很容易导致后代的基因丢失。
Prior to generating a new mouse model, it is important to plan the method that will be used to detect which of the mice generated have the mutation(s) desired. Nearly, all types of mutations may be detected using PCR. However, the choice of primers will differ depending upon the method used to generate the model. Transgenic mice should be genotyped across a unique junction fragment. Targeted ES cells used to generate knock-out or knock-in mice should be genotyped using primers from a unique marker in the construct and a region outside of the construct. Targeting in ES cells can also be detected using a genomic Southern blot. Mice targeted using CRISPR/Cas9 should have the region of interest amplified using PCR, and then be assessed for size changes (for large changes in sequence) by Surveyor Assay (for gene knock-out and point mutations) and/or sequenced to verify the mutation. Each of these models has a unique requirement for genotyping, and failure to understand the requirements can easily lead to loss of the gene in subsequent generations.