Fig 1.
Strategy for conditional knock-in generation via inversion.
(A) Scheme of the plasmid used to test recombination and stable inversion with lox71 and loxKR3. Magenta arrowhead: lox71; light blue arrowhead: loxKR3; grey arrowhead: loxP; red arrowhead: double mutant lox; green arrowhead: FRT site. (B) Restriction fragment analysis with BstBI of the plasmid (4.8 kb) after transformation in Cre-expressing E.coli. The correctly inverted plasmid results in restriction fragments of 3.5 kb and 1.3 kb. Lanes 1, 3–6: positive clones; lane 2: blank (water); MW: DNA molecular weight marker. (C) Two versions of the targeted exon (wild-type and mutated) are inserted in head-to-head orientation between a loxKR3 and a lox71 site. The two versions of the exon are separated by an FRT-flanked neomycin cassette. After targeting the neomycin cassette is deleted via Flp-mediated recombination in vivo. The mutation is activated by stable inversion of the two exons mediated by Cre-recombinase. The targeted exon in the Impad1 conditional knock-in is exon 2, as shown in the figure, while exon 7 is the targeted exon in the Clcn7 conditional knock-in. The sequences of lox sites used in this study are reported and mutated sequences are indicated by bold and are underlined. Lox71 carries mutations in the left element, while loxKR3 carries mutations in the right element.
Fig 2.
Generation of the Impad1 and Clcn7 mouse lines.
(A) Schematic drawing of the wild-type and targeted loci for Impad1. EcoRV restriction sites and fragments for Southern blot analyses are indicated in light blue. (B) Schematic drawings of the wild-type and targeted loci for Clcn7. EcoRI restriction sites and fragments for Southern blot analyses are indicated in light blue. Exons, indicated by arrowheads, are not numbered to simplify the figure. (C) Long range PCR analysis for correct homologous recombination of ES clones with primers SY08.19 and LRPCRneo1. The expected fragment of 7.1 kb confirms correct homologous recombination in the Impad1 locus. (D) Southern blot analysis of Impad1 targeted ES cells after EcoRV digestion using a neomycin specific probe; the 15.2 kb fragment corresponding to correct homologous recombination is indicated. (E) Genotyping PCR analysis for heterozygous Impad1 floxed pups using primers SY08.20 and SY08.21 flanking the lox71 site. The wild-type and floxed allele result in PCR products of 459 bp and 506 bp, respectively. (F) Long range PCR analysis for correct homologous recombination of ES clones with primers SY03.9 and LRPCRneo1. The expected amplicon of 5.8 kb confirms correct homologous recombination in the Clcn7 locus. (G) Southern blot analysis of Clcn7 targeted ES cells after EcoRI digestion using a neomycin specific probe; the 11.9 kb fragment corresponding to correct homologous recombination is indicated. (H) Genotyping PCR analysis for heterozygous Clcn7 floxed pups using primers SY03.11 and SY03.12 flanking the lox71 site. The wild-type and floxed allele result in PCR products of 539 bp and 588 bp, respectively. Black arrowhead: exon; green arrowhead: FRT site; light blue arrowhead: lox71 or loxKR3 site; red arrowhead: targeted exon 2 in Impad1 or targeted exon 7 in Clcn7; green arrow: neomycin cassette; SA: short arm; LA: long arm; E#: exon number; MW: DNA molecular weight marker.
Fig 3.
Phenotypic characterization of the Impad1D175N/D175N mouse.
(A) Gross morphology of wild-type and Impad1D175N/D175N newborn mice. Mutant animals die at birth and are considerably smaller than wild-type littermates demonstrating severe growth retardation. (B) X-rays of wild-type and mutant mice at birth. The mutant shows skeletal defects and growth retardation. (C) In Impad1D175N/D175N mice cleft palate is observed (arrow). Scale bars: 2 mm. (D) Alcian blue and alizarin red skeletal staining of Impad1D175N/D175N and wild-type bones at birth; the femur, tibia and fibula are markedly shorter compared to wild-type animals; rib cages of mutant mice display skeletal defects characterized by reduced sternal length and diminished rib spacing. Scale bars: 2 mm.
Fig 4.
Sulfation of chondroitin sulfate proteoglycans from femoral head cartilage.
Sulfation of proteoglycans was determined by HPLC disaccharide analysis after digestion by chondroitinase ABC and ACII of chondroitin sulfate proteoglycans from the femoral head cartilage of wild-type (WT) and Impad1D175N/D175N mice at birth. In parallel the same analysis was performed also in the Impad1 knock-out (Impad1-/-) mouse studied by Frederick [19]. The amount of non sulfated disaccharide (ΔDi-0S) relative to the total amount of disaccharides (ΔDi-0S, ΔDi-4S and ΔDi-6S) is significantly increased in mutant mice compared to the wild-types indicating proteoglycan undersulfation. Interestingly, the level of proteoglycan undersulfation in mutants is similar to the Frederick’s knock-out mouse. Three mice per group were used; data are reported as mean ± SD (Student’s t-test, *p<0.05; ***p<0.001).
Fig 5.
Relative expression analysis of the Impad1 gene.
RT-qPCR on total RNA isolated from skin of wild-type (WT), heterozygous (Impad1WT/D175N) and Impad1D175N/D175N newborn mice was performed with primers spanning exon 2 and exon 3. Expression of Impad1 mRNA normalized to Gapdh is absent in homozygous mutant animals compared to wild-types. Three mice per genotype were used; each sample was run in triplicate and three different experiments were performed.
Fig 6.
Alternative splicing analysis of Impad1 mRNA.
(A) RT-PCR using primers Imp7 and Imp12 that amplify a region spanning exon 1 to exon 5 was performed from skin total RNA of wild-type and Impad1D175N/D175N newborn mice and analysed by 1.5% agarose gel. In wild-type animals one band, 639 bp long, corresponding to the correctly spliced Impad1 transcript including the 5 coding exons is present. This band is not detected in mutant mice, conversely two different bands, 477 bp and 383 bp long, respectively are observed. (B) Sequencing of the two bands demonstrates that the two transcript variants lack exon 2 or both exon 2 and exon 3.
Fig 7.
Survival rate of Clcn7Flox/Flox mice.
Survival rates were calculated following Clcn7WT/WT (black line), Clcn7Flox/WT (blue line) and Clcn7Flox/Flox (red line) mice for 60 days. The Clcn7WT/WT and Clcn7Flox/WT survival rates overlap, for this reason the blue line is not visible in the graph. Survival rate of Clcn7Flox/Flox mice is dramatically reduced. Data are the mean ± SD of 5 mice for each group (Mantel-Cox test).
Fig 8.
Clcn7Flox/Flox mouse phenotype.
Twenty-one day old Clcn7WT/WT, Clcn7Flox/WT and Clcn7Flox/Flox mice were evaluated for (A) gross appearance and (B) tooth eruption. Clcn7Flox/Flox mice show reduced skeletal growth and no tooth eruption (the red arrow points to the absence of teeth). Scale bar: 1 cm.
Fig 9.
Bone phenotype of the Clcn7Flox/Flox mice.
Twenty-one day old (A) Clcn7WT/WT and (B) Clcn7Flox/Flox mice were sacrificed and then X-ray and μCT imaging were performed on femurs. A severe osteopetrotic phenotype with high bone mass is present in mutant mice. Pictures are representative of 5 mice per group.
Fig 10.
Alternative splicing analysis of Clcn7 mRNA.
(A) RT-PCR using primers that amplify a region spanning exon 6 to exon 25 was performed from bone total RNA of wild-type (Clcn7WT/WT) and Clcn7Flox/Flox mice and analysed by 1.5% agarose gel. In wild-type animals one band, 1683 bp long, corresponding to the correctly spliced Clcn7 transcript including 20 coding exons is present. This band is not detected in mutant mice, conversely a 1216 bp long band lacking exon 7–11 is observed.