One-Step Genotyping Method in loxP-Based Conditional Knockout Mice Generated by CRISPR-Cas9 Technology

Abstract

AbstractWith the development of CRISPR-Cas9 gene editing and in vitro fertilization (IVF) technology, we can now easily construct genetically modified mouse strains with indels, especially for loxP-based strategy. However, the general genotyping methods are time-consuming and unreliable given the loxP site is only 34 bp long. Here, based on the tetra primer-paired PCR amplification, we describe an efficient genotyping method which can simultaneously generate the internal control band, wild type (wt)-genotype band, and/or loxP-genotype band through one single PCR amplification. It is easy to interpret the mouse genotypes from the pattern of the bands. Further, the results could also help to exclude the possibility of minor cross-contamination, since the ratio between the bands’ quantity in wt/wt, wt/loxP, and loxP/loxP mice are relatively constant, which makes the genotyping more reliable when it is performed in a large amount.