Male germline recombination of a conditional allele by the widely used Dermo1-cre (Twist2-cre) transgene

Abstract

Conditional gene knockout using the Cre/loxP system is instrumental in advancing our understanding of the function of genes in a wide range of disciplines. It is becoming increasingly apparent in the literature that recombination mediated by some Cre transgenes can occur in unexpected tissues. Dermo1-Cre (Twist2-Cre) has been widely used to target skeletal lineage cells as well as other mesoderm-derived cells. Here we report that Dermo1-Cre exhibits spontaneous male germline recombination activity leading to a Cre-mediated recombination of a floxed Ptk2 (Protein tyrosine kinase 2, also known as Fak [Focal adhesion kinase]) allele but not a floxed Rb1cc1 (RB1 inducible coiled-coil 1, also known as Fip200 [FAK-family Interacting Protein of 200 kDa]) allele at high frequency. This ectopic germline activity of Dermo1-Cre occurred in all or none manner in a given litter. We demonstrated that the occurrence of germline recombination activity of Dermo1-Cre transgene can be avoided by using female mice as parental Dermo1-Cre carriers.

Keywords: Cre-loxP; Dermo1-Cre; Fak; Fip200; Twist2-Cre; conditional knockout; germline.

Figure 1. Fak floxed locus and rearrangement

(a) Schematic of mouse Fak gene and targeted allele. Fak gene is composed of FERM, Kinase, PXXP, and FAT domains from N terminal to C terminal. Exon3 locating at FERM domain was flanked by LoxP sites. Primers used to identify the wild type (WT), floxed (Flox) and rearranged (FAK^R) alleles are shown as solid arrows: primer pair P1/P2 amplifies a 800-bp WT and 1000-bp Flox bands; primer pair P1/P3 amplifies a 550-bp rearranged floxed Fak allele (FAK^R). (b) Genomic DNA was extracted from mouse tail and analyzed by PCR using P1, P2, and P3 primers to distinguish different types of alleles with or without the rearrangement of Fak floxed allele.

Figure 2. Unexpected Fak allele recombination in the absence of Dermo1-Cre transgene

(a) Schematic showing breeding strategies and Fak gene deletion using male mice as paternal Dermo1-Cre carrier to generate Fak conditional knockout mice. Male mice heterozygous for both the Fak floxed allele and the Dermo1-Cre transgene were mated with female mice homozygous for Fak floxed allele. Offspring exhibiting Fak deletion without Dermo1-Cre transgene was observed (indicated by broken line). The table below the scheme shows the expected as well as the observed genotype distribution. The numbers of animals per total number of animals (n=121) is shown in parentheses. (b) Representative PCR reaction showing genotyping result of the mice whose genotype showed the presence of Fak floxed allele and absence of both wild type allele and Dermo1-Cre transgene. Primer pair Cre 1/Cre 2 was used to amplify Dermo1-Cre transgene as a 696-bp band and Primer pair Alk2-5/Alk2-3 was used to amplify Alk2 gene as an internal DNA control (upper panel). Primer pair P1/P2 was used to amplify the floxed Fak allele (Flox) as 1000-bp band (middle panel). Primer pair P1/P3 was used to amplify a 550-bp rearranged floxed Fak allele (FAK^R) (lower panel). Asterisks at the bottom of the gel indicate progeny with unexpected rearrangement of Fak floxed allele (FAK^R).

Figure 3. Schematic showing breeding strategies to determine whether the occurrence of Fak floxed allele recombination is at germline level

(a) Female FAK^F/R mice were mated with male wild type mice. The table below the scheme shows the observed genotype distribution. The numbers of animals per total number of animals (n=45) is shown in parentheses. (b) Representative PCR reaction showing genotyping result of the offspring of female FAK^F/^R mice and male wild type mice. Primer pair P1/P2 was used to amplify WT and floxed Fak allele (Flox) as 800-bp and 1000-bp bands, respectively. Primer pair P1/P3 was used to amplify a 550-bp rearranged floxed Fak allele (FAK^R).

Figure 4. Schematic showing breeding strategies to determine whether the occurrence of Fak floxed allele recombination is at or after zygote stage

Male mice heterozygous for Dermo1-Cre transgene were mated with female mice homozygous for Fak floxed allele. The table below the scheme shows the expected as well as the observed genotype distribution. The numbers of animals per total number of animals (n=26) is shown in parentheses.

Figure 5. Schematic showing breeding strategies and Fak gene deletion using female mice as Dermo1-Cre carrier

Female mice heterozygous for both the Fak floxed allele and the Dermo1-Cre transgene were mated with male mice homozygous for Fak floxed allele. The table below the scheme shows the expected as well as the observed genotype distribution. The numbers of animals per total number of animals (n=123) is shown in parentheses. The data were obtained from 9 different female breeders.