Targeting genes for self-excision in the germ line

Abstract

A procedure is described that directs the self-induced deletion of DNA sequences as they pass through the male germ line of mice. The testes-specific promoter from the angiotensin-converting enzyme gene was used to drive expression of the Cre-recombinase gene. Cre was linked to the selectable marker Neor, and the two genes flanked with loxP elements. This cassette was targeted to the Hoxa3 gene in mouse ES cells that were in turn used to generate chimeric mice. In these chimeras, somatic cells derived from the ES cells retained the cassette, but self-excision occurred in all ES-cell-derived sperm.

Figure 1

Testes-specific self-excision. (A) A selectable marker gene, Neo^r, with a constitutive promoter, is transferred by homologous recombination to a specific locus in a mouse ES cell. The Neo^r gene is linked to a Cre gene that is under transcriptional control of the tACE promoter, and the two genes are flanked with loxP sites (P); the entire cassette, ACN, is introduced by gene targeting to a specific locus in a mouse ES cell. (B) ES cells, heterozygous for an allele containing the integrated cassette, are injected into wild-type mouse blastocysts and the blastocysts allowed to develop; the resulting animals are chimeric for wild-type (host-derived) cells (white) and ES-derived cells (black). (C) Male chimeric animals will transmit through their sperm one of two alleles of the locus of interest: wild-type (white) or mutant (gray); if self-excision has occurred the mutant allele will be marked only by a loxP site, the final product of the testes-specific self-excision reaction.

Figure 2

Targeting of a self-excision cassette to Hoxa3. (A) Self-excision cassette, ACN: The testes-specific elements from the mouse ACE gene (black arrow) are placed 5′ of the modified Cre structural gene (Gu et al. 1993) (red), followed, 3′, with the minimal polyadenylation signal from HSV–TK (Thomas and Capecchi 1987) (white box); an intron, derived from the SV40 t-antigen gene (white box), is inserted into the Cre gene; the Neo^r gene (blue) is controlled by a promoter from the mouse RNA polymerase II gene (black arrow) and followed also by the HSV–TK poly(A) site (white box). The 5′ and 3′ ends of this cassette contain loxP sites (P). (B) Gene targeting at Hoxa3: (Top line) The targeting vector pRVa3^ACN; the vector contains 11 kb of mouse genomic DNA into which the self-excision cassette ACN has been inserted in the homeodomain of Hoxa3 (McGinnis et al. 1984); the genomic sequences are linked to the HSV–TK gene (dark gray) and all are contained on a pUC-based plasmid backbone (light gray); the ACN cassette contains at its 5′ end an SstI site (S), used as a marker for homologous integration of the cassette at the Hoxa3 gene; (second line) the wild-type Hoxa3 locus; (bottom line) the predicted structure of the recombinant Hoxa3^ACN allele. The 5′ flanking probe used to detect recombination is indicated, and the diagnostic SstI-generated DNA fragments delineated beneath each locus. Yellow boxes designate Hoxa3 exons; other SstI sites in the vector are not indicated. (C) Southern transfer analysis: DNA from the parental cell line (ES) and the homologous recombinant ES lines used to generate mice was restricted with SstI; radiolabeled DNA probe is depicted in B.

Figure 3

Genetic transmission of Hoxa3 alleles. (A) The PCR-based genotyping of the three Hoxa3 alleles: Primer 1 (p1) is from the Hoxa3 intron; primer 2 (p2) is from coding exon 2-derived sequences (antisense); primer 3 (p3) is from the Neo^r gene. Predicted sizes are indicated; color coding is as in Fig. 2. (B) Genotyping of DNA: DNA is from wild-type ES cells (ES), recombinant ES cell line, 1h-9, tail biopsies from a chimeric male, χ3227, generated from 1h-9, and tail tissue from F₁ progeny of the chimera; amplified DNA was electrophoresed through agarose and stained with ethidium bromide. Sizes correspond to those listed in a. (C) Absence of excision in somatic tissue. A single chimeric male derived from cell line 1h-9 was sacrificed at 8 weeks of age. DNA extracted from each of the indicated tissues was analyzed by PCR as in B. Analysis of a second chimera showed an identical result.

Figure 3

Genetic transmission of Hoxa3 alleles. (A) The PCR-based genotyping of the three Hoxa3 alleles: Primer 1 (p1) is from the Hoxa3 intron; primer 2 (p2) is from coding exon 2-derived sequences (antisense); primer 3 (p3) is from the Neo^r gene. Predicted sizes are indicated; color coding is as in Fig. 2. (B) Genotyping of DNA: DNA is from wild-type ES cells (ES), recombinant ES cell line, 1h-9, tail biopsies from a chimeric male, χ3227, generated from 1h-9, and tail tissue from F₁ progeny of the chimera; amplified DNA was electrophoresed through agarose and stained with ethidium bromide. Sizes correspond to those listed in a. (C) Absence of excision in somatic tissue. A single chimeric male derived from cell line 1h-9 was sacrificed at 8 weeks of age. DNA extracted from each of the indicated tissues was analyzed by PCR as in B. Analysis of a second chimera showed an identical result.

Figure 3

Genetic transmission of Hoxa3 alleles. (A) The PCR-based genotyping of the three Hoxa3 alleles: Primer 1 (p1) is from the Hoxa3 intron; primer 2 (p2) is from coding exon 2-derived sequences (antisense); primer 3 (p3) is from the Neo^r gene. Predicted sizes are indicated; color coding is as in Fig. 2. (B) Genotyping of DNA: DNA is from wild-type ES cells (ES), recombinant ES cell line, 1h-9, tail biopsies from a chimeric male, χ3227, generated from 1h-9, and tail tissue from F₁ progeny of the chimera; amplified DNA was electrophoresed through agarose and stained with ethidium bromide. Sizes correspond to those listed in a. (C) Absence of excision in somatic tissue. A single chimeric male derived from cell line 1h-9 was sacrificed at 8 weeks of age. DNA extracted from each of the indicated tissues was analyzed by PCR as in B. Analysis of a second chimera showed an identical result.