Protamine-Cre recombinase transgenes efficiently recombine target sequences in the male germ line of mice, but not in embryonic stem cells - PubMed (original) (raw)

Protamine-Cre recombinase transgenes efficiently recombine target sequences in the male germ line of mice, but not in embryonic stem cells

S O'Gorman et al. Proc Natl Acad Sci U S A. 1997.

Abstract

The production of subtle or conditional mutations in mice through the combined use of site-specific and homologous recombination has become an increasingly widespread experimental paradigm in mammalian genetics. Embryonic stem cells containing recombinase transgenes that were expressed in the male germ line, but not in other tissues or in the embryonic stem cells themselves, would substantially simplify the production of such alleles. Here we show that transgenes comprised of the mouse protamine 1 promoter and the Cre recombinase coding sequence mediate the efficient recombination of a Cre target transgene in the male germ line, but not in other tissues. Embryonic stem cell lines generated from one of these transgenic strains were transfected with targeting vectors that included loxP-flanked selectable markers, and homologously recombined alleles containing the marker and functional loxP sites were isolated. These results establish the potential of the system for substantially reducing the time, effort, and resources required to produce homologously recombined alleles in mice that have been secondarily rearranged by a site-specific recombinase.

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Figures

Figure 1

Figure 1

Schematic of P2Bc and P2Br alleles. Cre-mediated recombination of the P2Bc allele results in the deletion of the neo cassette (Neo) of P2Bc that is flanked by two loxP sites, leaving a single loxP site and fusing the β-gal coding sequence to the initial codons of the RNA polymerase II coding sequence (solid boxes). Recombination increases the size of a _Pst_I fragment recognized by the RP2 probe, which is external to the targeting vector used, indicated by the shaded box below each allele. Positions of the PCR primers used (5′P and 3′P) are indicated on the diagrams of the P2Bc and P2Br alleles.

Figure 2

Figure 2

Southern blot of _Pst_I-digested tail biopsy DNAs from a +/P2Bc, +/PrmCre male (sire) and four of his progeny by a wild-type female probed with an RP2 probe (Upper) and then reprobed with a Cre probe (Lower). All RP2 mutant alleles in the progeny shown (lanes 1–3) were P2Br, and some progeny (no. 3 in this example) inherit a P2Br allele without inheriting a PrmCre transgene. Mouse 4 does not contain a PrmCre transgene and is homozygous wild-type at the RP2 locus.

Figure 3

Figure 3

Recombination in the testes of PrmCre mice. Southern blot of _Pst_I-digested DNA from testes (T) and one other tissue (K, kidney; B, brain; S, spleen) of males heterozygous for one of four PrmCre transgenes (58, 70, 71, 78) and the P2Bc allele. (Upper) Hybridization with an RP2 probe (see Fig. 1). Testis DNA from each male shows a P2Br allele signal, in addition to those generated by the wild-type RP2 (WT) and P2Bc alleles. Other tissues show only the WT and P2Bc signals. (Lower) Rehybridization with a Cre probe. The copy number of PrmCre transgenes (above each pair of lanes) varied among lines showing similar levels of recombination in testis.

Figure 4

Figure 4

Southern blot of PCR amplification products of the P2Br allele using tissues from a male heterozygous for the strain 71 PrmCre transgene and the P2Bc allele. DNA from 10 different tissues was amplified by using primers and conditions that produced a 350-bp product from the recombined, P2Br allele (see Fig. 1 and Materials and Methods). Each lane contains 10% of the reactions, except for the testis reactions, which were diluted 500- (T5), 250- (T2), and 100- (T1) fold before loading, and a liver reconstruction control (C), which was diluted 1:100 before loading. (Li, liver; Sp, spleen; Pa, pancreas; Th, thymus; Lu, lung; Ki, kidney; He, heart; Mu, skeletal muscle; Br, brain; No, no DNA control).

Figure 5

Figure 5

Targeting of the hoxb-1 locus in PrmCre ES cells by using a targeting vector that contains a loxP-flanked selectable marker. (A Top) Schematic of the wild-type hoxb-1 locus showing the positions of the two exons (open boxes), the position of a 5′ _Nru_I site and flanking _Bam_HI restriction endonuclease sites, and PCR primers (triangles) that amplify a 204-bp product from the wild-type allele that includes the _Nru_I site. (Middle) Predicted organization of homologously recombined hoxb-1 allele in which a neo cassette (NEO), flanked by loxP sites (L), has been inserted into the _Nru_I site shown in Top. The insertion creates a novel _Bam_HI site and the same PCR primers now amplify a 1,600-bp product. (Bottom) Predicted structure of the recombined allele shown in Middle after Cre-mediated excision of the neo cassette to leave a single loxP site in place of the _Nru_I site of the wild-type allele. Amplification with the same primers now yields a 268-bp product. (B) Southern blot of _Bam_HI-digested genomic DNAs harvested from a 96-well plate from 10 doubly selected (31) ES cell clones and hybridized with a probe (A) that is external to the targeting construct. All samples show the 7.5-kb band from the wild-type allele and four clones (∗) additionally show the 6-kb band predicted to result from homologous recombination. (C) PCR products from amplification of five homologously recombined PC3 ES cell clones (lanes 1–10) and the parental PC3 cell line (lane 11) by using the primers shown in A. The cells were either mock transfected (lanes 1–5) or transiently transfected with pOG231 (lanes 6–11). The recombinant clones and parental cell lines show the 204-bp amplification product of the wild-type allele, and the recombinant clones additionally show a 1,600-bp product (1600) resulting from amplification across the neo cassette and a nonspecific 1,100-bp amplification product (NS). The pOG231-transfected recombinant clones show an additional 268-bp product signaling the Cre-mediated excision of the neo cassette from the recombinant alleles of some cells.

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