Nucleic Acids Research

Figure 1. (A) Schematic representation of the dormant-hyg^R and active-hyg^R alleles. In the dormant-hyg^R cassette, the PGK-promoter is separated from the hyg^R gene by PGKneo^R flanked by two loxP repeats. Upon activity of the Cre recombinase the PGKneo^R cassette is excised and the PGK-promoter and the hyg^R gene are placed together resulting in an active-hyg^R allele. PCR primers were chosen in the PGK-promoter (PGK2-5[prime], CGC GAA GGT CCT CCG GAG CCC G, solid box) and the hyg^R gene (hygro4-3[prime], CGG CAC TTT GCA TCG GCC GCG C, hatched box). The expected PCR product prior to Cre activity is 2.2 kb (open arrow) and 0.36 kb (closed arrow) following Cre mediated recombination. To detect the presence of the floxed PGKneo^R cassette, a PCR assay was performed using a specific primer binding to the neo^R gene (o-neo-3, CCT GTC ATC TCA CCT TGC TCC TGC C, open box) and the primer hygro4-3[prime] under the conditions described. The expected size of the fragment dh-internal after amplification of the dormant-hyg^R cassette is 1.3 kb, while there is no PCR product if the active-hyg^R allele is present in the samples. PCR fragments are represented by double bars (=). (B) PCR based detection of Cre mediated recombination in transgenic mice. To show that the dormant-hyg^R cassette can be recombined in vivo, mice carrying the dormant-hyg^R cassette alone or dh4/cre littermates harboring the dormant-hyg^R cassette and a ubiquitously expressed Cre transgene were sacrificed 1 week after birth. Genomic DNAs from heart (H), liver (L), small intestine (I), skeletal muscle (Sk), kidney (K), spleen (S) and brain (B) were isolated and analyzed by PCR (30 cycles, 30 s at 94°C, 30 s at 69°C and 2 min at 72°C). The resulting PCR products had the correct sizes in samples derived from dh4 mice (open arrow) and in samples obtained from dh4/cre mice showing successful recombination of the dormant-hyg^R cassette (solid arrow). PCR controls are indicated as h (H₂O), a (active-hyg^R allele), d (dormant-hyg^R allele) and as [phis] (DNA molecular weight marker [phis]X174 HinfI, Gibco BRL).

A selection cassette, named dormant-hyg^R, was constructed (Fig. 1A). The dormant-hyg^R cassette contains the neo^R and hyg^R genes which confer resistance to G418 and hygromycin, respectively. The hyg^R gene is separated from the constitutive murine 3-phosphoglycerate kinase (PGK) promoter by PGKneo^R, flanked by two loxP repeats. In cells expressing the Cre recombinase, PGKneo^R is excised thereby placing the PGK-promoter and the hyg^R gene together, resulting in an active-hyg^R allele (Fig. 1A). To test whether the hyg^R gene is dormant prior to Cre recombinase activity, we transfected the dormant-hyg^R cassette either alone or together with the Cre recombinase expression vector pMCcre (4) into ES cells. After hygromycin selection, drug-resistant clones were obtained only in double transfected cells but not in cells carrying the dormant-hyg^R cassette, demonstrating that the hyg^R gene is silent without Cre activity (data not shown).

Mice harboring the dormant-hyg^R cassette were generated by zygote injection. Four transgenic founders were identified and one line, named dh4, was randomly chosen and kept for further analysis. To test if the dormant-hyg^R cassette is recombined in vivo we mated dh4 mice with a Cre recombinase transgenic mouse line, named cre, expressing the enzyme ubiquitously (K. Zeh and H. Baribault, unpublished results). In vivo recombination of the dormant-hyg^R cassette in the progenies was confirmed by PCR analysis. Genomic DNAs isolated from heart, liver, small intestine, skeletal muscle, kidney, spleen and brain were amplified using primers for the PGK-promoter and the hyg^R gene. The predicted PCR product for the dormant-hyg^R allele is 2.2 kb, while the amplified fragment for the active-hyg^R allele is 0.36 kb (Fig. 1A and B). Analysis of the PCR showed that Cre mediated recombination was found in all tissues of double transgenic mice confirming that the dormant-hyg^R cassette can be recombined in vivo (Fig. 1B).

To demonstrate that a Cre induced active-hyg^R allele provides hygromycin resistance in primary cell cultures derived from transgenic mice, dh4 and Cre recombinase mice (dh4/cre) were mated and embryos collected between day E14 and E17 of gestation. Embryonic fibroblasts (MEFs) were isolated from dh4 and dh4/cre embryos and subjected to hygromycin selection. MEFs from dh4 embryos grew in non-selective media (Fig. 2A) but were sensitive to hygromycin at a concentration of 0.1 mg/ml (Fig. 2B). MEFs isolated from dh4/cre embryos also grew in non-selective media (Fig. 2C) but in contrast were resistant to 0.1 mg/ml hygromycin (Fig. 2D) and tolerated doses of hygromycin up to 1 mg/ml (data not shown). This result shows that cells expressing the Cre recombinase can be selected from a heterogeneous cell population based on the activation of the dormant-hyg^R cassette.

Figure 2. Derivation of primary fibroblast cell cultures from transgenic embryos. dh4 and Cre recombinase mice were mated and embryos collected between gestation day E14 and E17. MEFs were isolated according to standard protocols (13). Double transgenic MEFs and control cells harboring the dormant-hyg^R cassette only were cultured in the presence of hygromycin at different concentrations (0.1, 0.25, 0.5, 0.75 and 1.0 mg/ml). After 7 days of selection the cultures were analyzed by light microscopy. The phase contrast picture shows MEFs established from dh4 embryos cultivated in theabsence (A) or in the presence (B) of 0.1 mg/ml hygromycin in the media.(C and D) MEFs cultures from dh4/cre embryos using the same conditions as described for (A) and (B), respectively.

To test whether an endogenous floxed allele is processed simultaneously with the dormant-hyg^R cassette, triple transgenic embryos were generated. These harbor, in addition to the dormant-hyg^R and the Cre recombinase transgenes, a floxed neo^R gene inserted by homologous recombination into exon 1 of the large subunit of the RNA Polymerase II gene. The targeted allele was named P2Bc (12). Upon Cre induced recombination, the neo^R gene is excised resulting in the recombined P2Br allele. The predicted PCR product for the unrecombined P2Bc allele is 1.7 kb while amplification of the recombined P2Br results in a fragment of 0.25 kb. MEFs isolated from two triple transgenic embryos (#23 and #31) were resistant to 0.1 mg/ml hygromycin in the media (data not shown). To study if both floxed alleles have been recombined successfully, genomic DNAs were isolated from these cell cultures before and after hygromycin selection. PCR analysis showed that only the active-hyg^R and the P2Br alleles were present (Fig. 3B, upper panels). To further confirm complete Cre mediated recombination, a specific PCR designed to detect the floxed DNA sequences within the dormant-hyg^R (dh-internal, Fig. 1A) and the P2Bc (P2Bc-internal, Fig. 3A) alleles was performed. Analysis showed that no amplification products were obtained indicating the absence of any floxed fragments in the MEFs (Fig. 3B, lower panels). These results clearly demonstrate that all hygromycin resistant cells underwent Cre mediated recombination of the dormant-hyg^R allele simultaneously with the endogenous floxed P2Bc allele.

Figure 3. (A) Schematic representation of the PCR products for the targeted P2Bc allele before (P2Bc) and after (P2Br) Cre induced recombination as well as for the control reaction, P2Bc-internal. The figure indicates the position of the upstream (P2Bc1-5[prime] CTT TCC CTC CTC CAG CCT TTC CCT CCC TAT, solid box) and the downstream (P2Bc2-3[prime] GCG TAA GGT TGA GGA CCC GAG CTT GGC ACT, hatched box) primers as well as primer o-neo-3 (Fig. 1A). The size of the unrecombined P2Bc allele is 1.7 kb (open arrow) and 0.25 kb for the recombined P2Br allele (closed arrow). The PCR was performed under the following conditions: 30 cycles, 30 s at 94°C, 30 s at 63°C and 2 min at 72°C. To amplify the P2Bc-internal fragment primer, P2Bc1-5[prime] was used together with primer o-neo-3 (28 cycles, 30 s at 94°C, 30 s at 68°C and 1 min at 72°C). Amplification results in a 0.8 kb fragment if the P2Bc allele is the template or in no PCR product if the P2Br allele is present. PCR products are indicated as (=). (B) Simultaneous recombination of the dormant-hyg^R cassette and the endogenous floxed allele by the Cre recombinase. Upper panels: a PCR assay was performed on genomic DNA isolated from MEFs, derived from triple transgenic embryos #23 and #31, before (-) and after (+) hygromycin selection. PCR products had the expected size for the recombined active-hyg^R allele (left) and the recombined P2Br allele (right), respectively. The additional product in the dormant-hyg^R PCR represents a background band detectable in all samples regardless of whether they harbor the dormant-hyg^R cassette or not (data not shown). Lower panels: genomic DNAs described above were amplified for the presence of the dh-internal (left, open arrow) or P2Bc-internal (right, closed arrow) fragments, respectively. No PCR products were detectable indicating the absence of the floxed DNA sequences. PCR controls: h (H₂O); left panel: a (active-hyg^R allele), d (dormant-hyg^R allele); right panel: r (P2Br allele), c (P2Bc allele). MW indicates molecular weight marker (1 kb ladder, Gibco BRL).

In conclusion, the method we present here describes the first system that allows to establish primary cell cultures with Cre induced somatic mutations from genetically altered mice by introducing the dormant-hyg^R cassette into either of the involved mouse lines.

ACKNOWLEDGEMENTS

We thank S. Kupriyanov and J. Avis (Mouse Genetics Facility at The Burnham Institute) for the production of transgenic mice, M. Bost and S. U. Stolley for excellent technical assistance and the personnel from the animal facility for animal care. We thank Dr A. Nagy and Prof. K. Rajewsky for providing the PGKneo^R cassette flanked by two loxP repeats and vector pMCcre, respectively. This work was supported by an AHA-CA Grant-in-Aid and BCRP-CA to H.B., and DFG and AHA-CA fellowships to K.Z.

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*To whom correspondence should be addressed. Tel: +1 619 455 6480; Fax: +1 619 646 3196; Email: kzeh@burnham-inst.org

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