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. Author manuscript; available in PMC: 2013 Feb 11.
Published in final edited form as: Genesis. 2008 Feb;46(2):87–91. doi: 10.1002/dvg.20370

Generation of a Conditional Null Allele for Dmp1 in Mouse

Jian Q Feng 1,*, Greg Scott 2, Dayong Guo 3, Baichun Jiang 1,6, Marie Harris 4, Toni Ward 2, Manas Ray 2, Lynda F Bonewald 3, Stephen E Harris 4, Yuji Mishina 5,2,*
PMCID: PMC3568775  NIHMSID: NIHMS87865  PMID: 18257058

Summary

Dentin matrix protein1 (DMP1), highly conserved in humans and mice, is highly expressed in teeth, the skeleton, and to a lesser extent in nonskeletal tissues such as brain, kidney, and salivary gland. Pathologically, DMP1 is associated with several forms of cancers and with tumor-induced osteomalacia. Conventional disruption of the murine Dmp1 gene results in defects in dentin in teeth and in the skeleton, including hypophosphatemic rickets, and abnormalities in phosphate homeostasis. Human DMP1 mutations are responsible for the condition known as autosomal recessive hypophosphatemic rickets. For better understanding of the roles of DMP1 in different tissues at different stages of development and in pathological conditions, we generated Dmp1 floxed mice in which loxP sites flank exon 6 that encodes for over 80% of DMP1 protein. We demonstrate that Cre-mediated recombination using Sox2-Cre, a Cre line expressed in epiblast during early embryogenesis, results in early deletion of the gene and protein. These homozygous Cre-recombined null mice display an identical phenotype to conventional null mice. This animal model will be useful to reveal distinct roles of DMP1 in different tissues at different ages.

Keywords: DMP1, Cre-loxP, gene targeting, mouse, bone

Cell type-specific gene ablation by Cre-loxP technology is particularly useful for determining the distinct tissue-specific function of a gene that has multiple functions and/or is lethal when deleted (Gu et al., 1993; Kwan, 2002). Dentin matrix protein1 (DMP1) belongs to the first category as DMP1 is expressed in several mineralized tissues such as dentin, cartilage, cementum, and bone (D’souza et al., 1997; Feng et al., 2002; Hirst et al., 1997; MacDougall et al., 1997). Interestingly, DMP1 is primarily expressed in osteoblasts during embryonic development and then mainly in osteocytes during postnatal development. More recently, DMP1 has been detected in nonmineralized tissues including brain, kidney, and salivary gland. Furthermore, DMP1 has been detected in the following cancer tissues: breast, uterus, colon, lung, and oral cavity (Chaplet et al., 2003; Fisher et al., 2004; Ogbureke and Fisher, 2004, 2005; Ogbureke et al., 2007; Terasawa et al., 2004; Toyosawa et al., 2004).

Conventional deletion of the Dmp1 allele in mice leads to abnormalities in mineralization, defects in maturation of osteoblasts and odontoblasts, as well as hypophosphatemic most likely due to increased fibroblast growth factor 23, a hormone secreted from osteoblasts/osteocytes and targeted to kidneys (Feng et al., 2006; Ling et al., 2005; Lu et al., 2007; Ye et al., 2004, 2005). Discoveries of DMP1 mutations in patients with autosomal recessive hypophosphatemia rickets not only shows the clinical relevance of DMP1 in human disease, but also extends the function of bone to that of an endocrine organ that regulates Pi homeostasis (Feng et al., 2006; Lorenz-Depiereux et al., 2006).

The key challenge has been to dissect out the function of DMP1 within the various tissues as listed above and the function of DMP1 at different times during development. This obviously cannot be accomplished by a simple knockout approach as preformed previously (Feng et al., 2003, 2006; Ling et al., 2005; Lu et al., 2007; Ye et al., 2004, 2005). To overcome this limitation, we generated conditional alleles of Dmp1 using Cre-loxP technology.

Generation of the Conditional Allele of Dmp1

Dmp1 gene consists of six exons and spans about 11-kb (AK132177, NCBI). To generate a conditional allele for Dmp1, we built a targeting vector that contained loxP sites to flox the exon 6 (Fig. 1a), the largest exon encoding over 80% of DMP1 amino acids. The 5′ loxP linked with multiple restriction sites was inserted into intron 5, and PstI was used for Southern blot confirmation. The 3′ loxP linked to a neo cassette (a selection marker) was inserted into 3′ side of exon 6. Note that we used FRT sites to flank the neo cassette for an option to remove the neo gene with flippase (Flp) recombinase.

FIG. 1.

FIG. 1

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(a) Targeting construct. Pgk-neo cassette flanked by FRT sites followed by a loxP site was inserted into Spe1 site in 3′ side of the exon 6. Another loxP site with a PstI site in it was inserted to an EcoRI site in the intron 5. Shaded areas in exons are protein coding regions. Positions of 5′ and 3′ external probes for Southern analyses are shown. B, BamHI; Hd, HindIII; RI, EcoRI, P, PstI; S, SpeI; Sm, SmaI. (b) Confirmation of the targeting event in ES cells. Genomic DNA from WT ES cells (lane 1) and correctly targeted cells (lane 2) were digested with enzymes and probes as indicated. PstI digestion with the 5′ probe confirmed the presence of loxP site in intron 5.

Confirmation of Deletion of Dmp1 Allele Using Cre-loxP Approach

Correct targeting events were confirmed by Southern blotting using 5′ and 3′ external probes, and the presence of 5′ loxP site was confirmed by a loxP-specific probe (Fig. 1b). The targeted allele was designated as the floxP + neo (fn) allele. After confirmation of germ-line transmission, F1 offspring heterozygous for fn were intercrossed to generate mice homozygous for fn. Resulted F2 showed expected ratio (+/+:fn/+:fn/fn = 27:56:22) of offspring. In addition, F1 offspring heterozygous for fn were bred with ROSA26-Flp mice, containing Flp recombinase, to remove the neo cassette through recombination at the FRT site. Removal of the neo was confirmed by Southern blotting using the 5′ external probe with BamHI or PstI digested genomic DNA to get a 16.5 kb or 7.5 band, respectively (Fig. 1a). The targeted allele without the neo cassette was named as floxP (fx) allele. Intercross between heterozygous mice for fx allele generated the expected ratio of the mice homozygous for fx (+/+:fx/+:fx/fx = 17:41:16). Furthermore, PCR strategy was developed as shown in Figure 2, which was able to differentiate wt allele, fn allele, Cre-recombined allele (designated as DE allele), and a targeted null allele that was reported earlier (Feng et al., 2003). To verify whether the loxP sites are functional, mice heterozygous for fn were bred with Sox2-Cre transgenic mice, a Cre targeted in the epiblast cells, to remove the floxed exon 6. Resulted mice were analyzed by Southern blot analysis and PCR for confirmation of Cre-dependent deletion as well as the efficiency of Cre recombination of the floxed allele in vivo (Fig. 2c).

FIG. 2.

FIG. 2

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PCR strategy and genotyping. (a) Schematic representation of structure of each allele and positions of primers. The conventional null allele replacing exon 6 by an IERS β-gal cassette (lacZ-knock in allele) is shown at the top. Primers A, B, and C are used to differentiate the conventional null allele from WT, whereas primers D and E are used to differentiate the fn allele from WT. Primers D and F are used to identify the Cre-recombined allele. All PCR shown in lanes 1 were produced using primers A, B, and C, whereas those shown in lanes 2 were produced using primers D, E, and F. (b) Genotyping results of the conditional allele. The primer set D and E differentiates fn alleles from WT (lane 2). (c) Genotyping results of the Cre-recombined allele along with the conventional null allele. Cre-recombination was detected by primers D and F (lane 2). All possible combinations of Dmp1 alleles can be differentiated by 2 PCR reactions as shown in lanes 1 and 2.

Similarities of Skeletal Phenotype in Both the Conventional Dmp1 Null and the Cre-Recombined Dmp1 Mice

For a better comparison, these heterozygous mice for Cre-recombined allele were intercrossed for obtaining mice homozygous for Cre-recombined Dmp1 allele (ΔE/ΔE). Furthermore, a second breeding group was set between heterozygous mice for the Cre-recombined allele (ΔE/+) and heterozygous mice for the conventional null allele (+/−) for obtaining transheterozygous null mice (ΔE/−). The phenotypes obtained from these two groups were compared to that in the conventional Dmp1 null mice (see below for details).

As is evident in Figure 3, homozygous mice for the conventional null allele, trans-heterozygous mice for the Cre-recombined and the conventional null allele, and homozygous mice for the Cre-recombined allele developed the similar skeletal morphological abnormalities at the age of 10 days, including significantly expanded metaphyses, defective maturation of woven bone into the lamellar bone, and delayed secondary ossification. Similar defects are also observed in both the Cre-recombined and conventional null mice at 2 months of age (see Fig. 4). The trans-heterozygous mice (ΔE/−) showed the same phenotype at both stages (data not shown). Mice heterozygous for the fn allele over the Cre-recombined allele (fn/ΔE) did not show any detectable changes at both stages (Figs. 3 and 4) indicating that the floxed allele of Dmp1 is functionally wild type even in the presence of the neo selection cassette. Growth plates stained with Safranin-O confirmed a ricketic phenotype (Fig. 5a), and immunohistochemical staining showed the absence of DMP1 in both the Cre-recombined and conventional null mice (Fig. 5b).

FIG. 3.

FIG. 3

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Cre-recombined Dmp1 null mice show similar skeletal abnormalities to those of conventional null, KO, mice at the age of 10 days. (a) Representative radiographs of skeletons from control (+/−), Dmp1 conventional null (−/−), Dmp1-fn allele over Cre-recombined allele (fn/ΔE) and Dmp1 Cre-recombined null (ΔE/ΔE) mice at the age of 10 days, showing shortened and malformed long bones (arrows), delayed epiphyses (open arrow) and expanded metaphyses (arrow head). (b) Quantitative analyses of areas of epiphyses and metaphyses obtained from femurs, showing a significant difference between KO or CKO and the control.

FIG. 4.

FIG. 4

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Cre-recombined Dmp1 null mice display a similar ricketic skeletal phenotype to conventional null, KO mice, including shorter long bones, wider midshafts, delayed epiphyses, and expanded metaphyses. (a) Representative radiographs of skeletons from control (+/−), Dmp1 conventional null (−/−), Dmp1-fn allele over Cre-recombined allele (fn/ΔE) and Dmp1 Cre-recombined null (ΔE/ΔE) mice at the age of 2 months. (b) Enlarged radiographs of hind limbs from 2 months animals. (c) Quantitative analyses of the length and width of femur.

FIG. 5.

FIG. 5

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Confirmation of a ricketic skeletal phenotype in mice with deletion of DMP1 conventionally and with Cre-recombination. (a) Representative sections from safranin-O stained growth plates from 2-month-old animals, showing a highly disrupted growth plate in both conventional and conditional KO (arrows). (b) DMP1 is highly expressed in WT osteocytes (arrows), and DMP1 protein cannot be detected using polyclonal DMP1 antibody in both conventional and Cre-recombined null long bones obtained from 10-day-old mice.

In conclusion, we have generated a conditional Dmp1 allele and shown that homozygous general deletion of exon 6 leads to absence of DMP1 protein, accompanied by typical rickets with delayed secondary ossification, enlarged growth plate with dramatic expansion of hypertrophic chondrocyte zone and short limbs. In the future, crossing this floxed line with transgenic mice that express Cre-recombinase in a tissue-specific manner will facilitate studies determining the distinct roles of DMP1 in different tissues where it is normally expressed, and allow determination of relative importance in mineralized as compared to nonmineralized tissues.

MATERIALS AND METHODS

Generation of a Dmp1-Floxed Allele

A 18 kb HindIII fragment, containing all introns and exons plus both 5′-and 3′-homologous arm regions, was isolated from a 129 BAC clone and used to construct a conditional gene targeting vector (Fig. 1a). Specifically, a 6.5-kb EcoRI 5′-fragment and a 6.5-kb EcoRI/SmaI 3′ fragment were subcloned into a bluescript SK vector separately. A Pgk-neo cassette flanked by FRT sites followed by a loxP site was introduced into a SpeI site in the 3′ side of exon 6 of Dmp1. A 5′ loxP site marked with a PstI site was introduced to an EcoRI site in intron 5. The position of the probes used for Southern analysis are shown in Figure 1a. The sizes of the restriction fragments detected by these probes in WT and targeted DNA are shown above the locus. Linearized targeting vector was electroporated into 1 × 107 AB2.2 ES cells (Lexicon Genetics, Woodlands, TX). Three hundred G418-resistant ES cell clones were initially screened by Southern blot and 12 correctly targeted ES cell clones were identified. Five of them were confirmed to possess the loxP site in intron 5 (fn/+). The ratio of these two types of clones we obtained without or with the loxP site is close to 1:1 because the loxP site in intron 5 divides the left homologous arm in about 1:1(Cheah and Behringer, 2000). The targeted ES clones were injected into blastocysts from C57BL/6 mice. The resulted chimeras were bred to C57BL/6 females and F1 agouti offspring were genotyped by Southern analyses. Four targeted clones were used for injection and three of them went through germline transmission.

Subsequently, mice heterozygous for Dmp1 floxed allele with a Pgk-neo cassette (fn/+) were bred with Flipper mice (Farley et al., 2000) to remove the neo cassette (fx/+). The fn/+ mice were also bred with Sox2-Cre mice (Hayashi et al., 2002) to delete exon 6 to generate recombined null allele (ΔE/+). All animal works were approved by NIH/NIEHS animal care and use committee.

Genotype Analyses

Genotypes were determined by Southern blot both with 5′ and 3′ external probes shown in Figure 1a. For PCR analyses, primers D and E were used to amplify fragments from wildtype (379 bp) and Dmp1 floxed alleles (494 bp for both fn and fx). Primers A and B were also used to amplify fragments from wildtype (379 bp) whereas primers A and C were used to detect null allele (230 bp) generated previously by knocking in a lacZ cassette (Feng et al., 2003). Primers D and E were used to detect Cre-dependent deletion of the floxed region (300 bp for ΔE). Primers; A (5′-CTTGACTTCAGGCAAATAGTGACC-3′), B (5′-GCGGAATTCGATAGCTTGGCTG-3′), C (5′-CTGTTCCTCACTCTCACTGTCC-3′), D (5′-GGCTCA TGTGTATAATTCCCAG-3′), E (5′-GCCTGAAGTCAAGGTA AACAG-3′), F (5′-TTCATGAGGTAATTTAAGAAAAGTCG-3′).

Acknowledgments

Contract grant sponsor: US National Institutes of Health; Contract grant numbers: AR051587, AR046798, AR046798, AR046798; Contract grant sponsor: Intramural Research Program of the NIH, National Institute of Environmental Health Sciences; Contract grant number: ES071003-10.

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