Genotyping Genetically Modified (GM) Mice
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.
Key wordsGenotyping Genetically engineered mice Transgenic Targeted Knock-out mice Knock-in mice CRISPR/Cas9 Germ line transmission Chimera Mosaic PCR
The authors would like to thank Dr. Vinod Pant for sharing his expertise with Southern blot analysis. This work was supported by a Cancer Center Support Grant NCI # CA016672(GEMF).
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