Cre-mediated germline mosaicism: a new transgenic mouse for the selective removal of residual markers from tri-lox conditional alleles

Abstract

The binary Cre-lox conditional knockout system requires an essential part of the target gene to be flanked by loxP sites, enabling excision in vivo upon Cre expression. LoxP sites are introduced by homologous recombination, together with a selectable marker. However, this marker can disturb gene expression and should be removed. The marker is therefore often prepared with a third, flanking loxP site (tri-lox construct), facilitating its selective removal by partial Cre-lox recombination. We have shown that this excision can be achieved in vivo in the germline using EIIaCre transgenic mice, and have described the advantages of in vivo over in vitro removal. We show here that MeuCre40, a new transgenic mouse, more reliably and reproducibly generates an optimal partial mosaic Cre-lox recombination pattern in the early embryo. This mosaicism was transmitted to the germline and to many other tissues. Alleles with partial deletions, in particular floxed alleles from which the selectable marker was removed, were readily recovered in the next generation, after segregation from the transgene. Segregation via paternal or maternal transmission led to successful recovery of the alleles of interest. We also obtained total deletion of the floxed regions in the same experiment, making this transgene a polyvalent Cre-lox tool. We rigorously tested the ability of MeuCre40 to solve tri-lox problems, by using it for the in vivo removal of neo(R)- and hprt-expression cassettes from three different tri-lox mutants.

Figure 1

Tri-lox alleles, transgene construction and breeding strategy. (A) Three tri-lox configurations concerning the genes encoding IGF-1R (a), APC (b) and Doublecortin (c) were tested. Triangles indicate loxP sites, white boxes indicate selectable expression cassettes and shaded boxes indicate exons (ex). The probe and restriction sites used for Southern analysis of the IGF-1Rtri-lox alleles are indicated; neo^R designates the neomycin resistance expression cassette, hprt designates the hypoxanthine phosphoribosyl transferase mini-gene (13). The I-SceI meganuclease site was introduced together with the loxP sites. Arrows indicate primers P1–P4 (see Materials and Methods for details). The size of the floxed exonic regions and of the floxed cassettes is indicated in kilobases without taking into account the small loxP insertions. (B) The DNA construct used to generate MeuCre40 had a total size of 4.3 kb. Cre, wild-type Cre cDNA; MP, minimal CMV promoter; p(A), polyadenylation signal; rtTA, reverse tetracycline-dependent transactivator (26); tetO, tetracycline resistance operon; arrows indicate transcription start sites; further details are provided in a previous paper (25). (C) Heterozygous F0 tri-lox mice were bred to obtain double transgenics in the F1 generation, all of which proved to be mosaic. The crossing of these mosaic mice with wild-type mice resulted in the offspring having a 50% probability of inheriting one of the several mutant alleles and a 50% probability of receiving MeuCre40. F2 animals were screened for MeuCre40 and mutant allele segregation. sel, selectable expression cassette; Δsel, deletion of the selectable marker neo^R or hprt; Δex, deletion of the floxed exon.

Figure 2

MeuCre40-induced Cre-lox recombination. (A) Scanned Southern blot analysis (double digest HincII/I-SceI; probe as indicated in Fig. 1A) of partial recombination in a MeuCre40/IGF-1Rtri-lox animal showed that Δneo alleles (1.7 kb) were present in considerable quantity compared with unrecombined IGF-1Rtri-lox alleles (3.5 kb) and the corresponding wild-type (WT) IGF-1R allele (2.4 kb). The profile generated by the related strain MeuCre48 showed a shift towards stronger recombination, with fewer Δneo alleles and a peak corresponding to total excisions (Δtotal; 0.8 kb). Signals indicating selective deletions of the floxed exon region (Δex; 2.6 kb) were superimposed over those of the wild-type (arrows), and could not be differentiated in this Southern blot analysis. Nevertheless, the partial Cre-lox excision pattern seen in APC and Doublecortin (data not shown) indicated that the final recombination profile depended to some degree on the particular interaction between MeuCre40 and the respective tri-lox alleles. (B) Partial Cre-lox recombination patterns were assayed by a multiplex PCR that detected relative amounts of unrecombined and recombined (Δtotal) IGF-1R alleles in the same reaction (28). The flox band and the WT band amplified with primer pair P3P4, the Δtotal band amplified with primer pair P1P4 (see Fig. 1A). Shown here for six representative MeuCre40/IGF-1Rflox animals, this test revealed little inter-individual variability, and most double transgenics displayed a recombination of 30–60% (bar graph, n = 25). (C) We observed only minor differences in the Cre-lox recombination pattern between major tissues. Male and female gonads (two lanes on the right) showed similar patterns of recombination to other tissues. (D) We investigated whether maternal (mat) MeuCre40 transmission affected the frequency of recombination, which was not the case (pat, paternal).

Figure 3

Histology of MeuCre40- and MeuCre48-induced loxP recombination. Newborn mice harboring MeuCre40 or MeuCre48 and a reporter gene indicating Cre-lox recombination by lacZ activity (29), were killed and tissue sections stained for lacZ activity. Shown here in six representative tissues, the patterns were clearly mosaic and very similar in the two strains. Black spots indicate Cre-lox recombinant cells. The positive control panels show lacZ activity in sections from newborns with a constitutively activated Rosa26 lacZ reporter gene. The negative control panels contain sections from mice with an unrecombined reporter gene, that produces no staining at all. Negative controls are recorded as phase contrast micrographs. Size bars indicate 20 µm. E, skin epithelium; H, hair follicle; L, intestinal lumen.

Figure 4

Developmental kinetics of MeuCre40-induced recombination in double transgenic (MeuCre40/IGF-1Rflox) animals. The percentage of recombined IGF-1Rflox reporter gene was determined from the eight-cell stage to the adult animal by a multiplex PCR assay (primers P1, P3 and P4; Fig. 1A) that detected relative amounts of unrecombined versus recombined allelic forms of the floxed IGF-1R gene (28). DNA was extracted from whole embryos, or postnatally from skin biopsies. Recombination was first observed during morula and blastocyst stages. Few changes were observed after E10. No Cre-lox recombination was detected in eight-cell embryos. Error bars indicate the SEM; total n = 52.