Introduction of loxP sites by electroporation in the mouse genome; a simple approach for conditional allele generation in complex targeting loci.

BACKGROUND: The discovery of the CRISPR-Cas9 system and its applicability in mammalian embryos has revolutionized the way we generate genetically engineered animal models. To date, models harbouring conditional alleles (i.e. two loxP sites flanking an exon or a critical DNA sequence of interest) are amongst the most widely requested project type that are challenging to generate as they require simultaneous cleavage of the genome using two guides in order to properly integrate the repair template. An approach, using embryo sequential electroporation has been reported in the literature to successfully introduce loxP sites on the same allele. Here, we describe a modification of this sequential electroporation procedure that demonstrated the production of conditional allele mouse models for eight different genes via one of two possible strategies: either by consecutive sequential electroporation (strategy A) or non-consecutive sequential electroporation (strategy B). This latest strategy originated from using the by-product produced when using consecutive sequential electroporation (i.e. mice with a single targeted loxP site) to complete the project. RESULTS: By using strategy A, we demonstrated successful generation of conditional allele models for three different genes (Icam1, Lox, and Sar1b), with targeting efficiencies varying between 5 and 13%. By using strategy B, we generated five conditional allele models (Loxl1, Pard6a, Pard6g, Clcf1, and Mapkapk5), with targeting efficiencies varying between 3 and 25%. CONCLUSION: Our modified electroporation-based approach, involving one of the two alternative strategies, allowed the production of conditional allele models for eight different genes via two different possible paths. This reproducible method will serve as another reliable approach in addition to other well-established methodologies in the literature for conditional allele mouse model generation.

Implementation of a PCR assay of Pasteurella pneumotropica to accurately screen for contaminated laboratory mice.

The authors implemented a PCR protocol to rapidly screen for Pasteurella pneumotropica and to accurately identify contaminated laboratory mice in a clinical setting. This protocol was implemented in response to a severe outbreak of P. pneumotropica in their animal facility. Although a sentinel program was in place to routinely screen for P. pneumotropica, it was inadequate for the identification of contaminated animals. As a result, several additional strains of mice were contaminated and developed clinical signs of infection. The authors implemented a screening method using PCR with reported primer pairs previously developed to identify the biotype isolates of P. pneumotropica in laboratory mice. Throat culture swabs were collected from live mice and placed in a bacterial culture. The DNA from these cultures was isolated and screened by PCR. This procedure enabled the authors to eliminate P. pneumotropica from several animal housing rooms. The assay can be easily applied in most animal facilities.