Genotyping Protocols for Genetically Engineered Mice

Abstract

Historically, the laboratory mouse has been the mammalian species of choice for studying gene function and for modeling diseases in humans. This was mainly due to their availability from mouse fanciers. In addition, their short generation time, small size, and minimal food consumption compared to that of larger mammals were definite advantages. This led to the establishment of large hubs for the development of genetically modified mouse models, such as the Jackson Laboratory. Initial research into inbred mouse strains in the early 1900s revolved around coat color genetics and cancer studies, but gene targeting in embryonic stem cells and the introduction of transgenes through pronuclear injection of a mouse zygote, along with current clustered regularly interspaced short palindromic repeat (CRISPR) RNA gene editing, have allowed easy manipulation of the mouse genome. Originally, to distribute a mouse model to other facilities, standard methods had to be developed to ensure that each modified mouse trait could be consistently identified no matter which laboratory requested it. The task of establishing uniform protocols became easier with the development of the polymerase chain reaction (PCR). This chapter will provide guidelines for identifying genetically modified mouse models, mainly using endpoint PCR. In addition, we will discuss strategies to identify genetically modified mouse models that have been established using newer gene‐editing technology such as CRISPR. Published 2023. This article is a U.S. Government work and is in the public domain in the USA.Basic Protocol 1: Digestion with proteinase K followed by purification of genomic DNA using phenol/chloroformAlternate Protocol: Digestion with proteinase K followed by crude isopropanol extraction of genomic DNA for tail biopsy and ear punch samplesBasic Protocol 2: Purification of genomic DNA using a semi‐automated systemBasic Protocol 3: Purification of genomic DNA from semen, blood, or buccal swabsBasic Protocol 4: Purification of genomic DNA from mouse blastocysts to assess CRISPR gene editingBasic Protocol 5: Routine endpoint‐PCR‐based genotyping using DNA polymerase and thermal cyclerBasic Protocol 6: T7E1/Surveyor assays to detect insertion or deletions following CRISPR editingBasic Protocol 7: Detecting off‐target mutations following CRISPR editingBasic Protocol 8: Detecting genomic sequence deletion after CRISPR editing using a pair of guide RNAsBasic Protocol 9: Detecting gene knock‐in events following CRISPR editingBasic Protocol 10: Screening of conditional knockout floxed mice