Genome Wide Conditional Mouse Knockout Resources

Abstract

The International Knockout Mouse Consortium (IKMC) developed high throughput gene trapping and gene targeting pipelines that produced mostly conditional mutations of more than 18,500 genes in C57BL/6N mouse embryonic stem (ES) cells which have been archived and are freely available to the research community as a frozen resource. From this unprecedented resource more than 6,000 mutant mouse strains have been produced by the IKMC and mostly the International Mouse Phenotyping Consortium (IMPC). In addition, a cre-driver resource was established including 250 inducible cre-driver mouse strains in a C57BL/6 background. Complementing the cre-driver resource, a collection of comprising 27 cre-driver rAAVs has also been produced. The resources can be easily accessed at the IKMC/IMPC web portal (www.mousephenotype.org). The IKMC/IMPC resource is a standardized reference library of mouse models with defined genetic backgrounds that enables the analysis of gene-disease associations in mice of different genetic makeup and should therefore have a major impact on biomedical research.

Figure 1:

Conditional gene inactivation by a SAbgeopA cassette. A. The SAbgeopA cassette flanked by recombinase target sites (RTs) in a FlEx configuration is illustrated after integration into an intron of an expressed gene. Transcripts (shown as gray arrows) initiated at the endogenous promoter are spliced from the splice donor (SD) of an endogenous exon (here exon 1) to the splice acceptor (SA) of the SAbgeopA cassette and terminate prematurely at the gene trap’s polyadenylation (pA) site thereby causing a mutation. Exposure to flpo inverts the SAbgeopA cassette onto the non-coding strand, which reactivates normal splicing. This repairs the mutation by restoring normal gene expression. A second, Cre mediated re-inversion positions the SAbgeopA cassette back onto the coding strand and re-induces the mutation. Arrows indicate primer positions within FlipRosabgeo used to diagnose inversions. Frt (yellow triangles) and F3 (green triangles), heterotypic target sequences for the flpe- and flpo-recombinases; loxP (purple triangles) and lox5171 (red triangles), heterotypic target sequences for the cre-recombinase; SA splice acceptor; bgeo, b-galactosidase-neomycin-phosphotransferase fusion gene; pA, polyadenylation sequence.

Figure 2:

IKMC gene targeting strategies. A) Knock-out first conditional ready targeting strategy. The targeting vector contains a splice-acceptor carrying lacZ reporter and neomycin selector cassette flanked by frt and loxP sites followed by a critical exon flanked by loxP site. After integration into the genome this allele is named tm1a allele. Exposure to flp recombinase deletes the reporter and selector cassette resulting in a wild type conditional floxed allele (tm1c). The critical exon can be deleted by cre recombinase expression (tm1d). Exposure of the tm1a allele first to cre recombinase deletes neomycin cassette and the critical exon leading to a reporter null allele (tm1b). B) Represents the generation of entire gene deletions using large BAC targeting vectors integrating a reporter and loxP flanked selector cassette.

Figure 2:

IKMC gene targeting strategies. A) Knock-out first conditional ready targeting strategy. The targeting vector contains a splice-acceptor carrying lacZ reporter and neomycin selector cassette flanked by frt and loxP sites followed by a critical exon flanked by loxP site. After integration into the genome this allele is named tm1a allele. Exposure to flp recombinase deletes the reporter and selector cassette resulting in a wild type conditional floxed allele (tm1c). The critical exon can be deleted by cre recombinase expression (tm1d). Exposure of the tm1a allele first to cre recombinase deletes neomycin cassette and the critical exon leading to a reporter null allele (tm1b). B) Represents the generation of entire gene deletions using large BAC targeting vectors integrating a reporter and loxP flanked selector cassette.

Figure 3:

EUCOMMTOOLS cre/ERT2 targeting vectors. The targeting vectors contain green fluorescent protein and cre/ERT2 separated with a T2A peptide sequence. The selection cassette is flanked with rox sites and is removable using dre recombinase.

Figure 4:

Cre-activity annotation of PbPrcap promoter driving cre-recombinase strain in the bone marrow (A), thymus (B), lymph node (C) and the peyer’s patch of the midgut (D) in P14 mice. The expression was detected in the entire lymphatic system from lymphoblastic precursors in the bone marrow to mature lymphocytes in the lymphatic nodes. Therefore, the inducible cre-driver system will allow gene inactivation at different stages of lymphocyte differentiation.

Figure 5:

Cre-activity of the TRpvl promoter cre-driver strain in Purkinje cells of the cerebellar cortex of P14 mice.