Figure 1.
Figure 1. Zfp521 favors bone formation in mature OBs. (A) Generation of null and conditional Zfp521 alleles. Zfp521 genomic region encoding exon 4 is shown. Restriction fragment sizes are indicated as well as the positions of the internal and the two flanking probes used for genotyping analysis (Roman numerals). The shaded area indicates the part of the genomic region included in the targeting vector, and the three different alleles are shown. “neo” is the result of the gene-targeting event. “cko” is the conditional knockout allele derived from the neo allele after Flpe-mediated excision of the PGK-neo cassette. One Frt site and two loxP sites remain in the locus. “ko” is the null allele derived from the “neo” or from the “cko” allele by Cre-mediated recombination between the two loxP sites. Only a single loxP site remains in the modified locus. Splicing of exon 3 to exon 5 generates a frameshift. loxP and Frt sites are indicated as closed and open triangles, respectively. Neo, PGK-em7-neomycin dual selection cassette for bacteria and embryonic stem cells; TK, thymidine kinase cassette for counter-selection in embryonic stem cells; X, XbaI; B, BamHI; N, NotI. The genomic region is not drawn to scale. (B) Results of a Southern blot analysis of BamHI-digested tail DNA, probed with the internal probe (III). (C) Northern blot analysis of whole-brain RNA from 3-wk-old mice using a full-length Zfp521 cDNA probe. The blot was rehybridized with a Gapdh probe as a control for RNA quality. wt, wild type; Δexon4, position of the residual mRNA after removal of exon 4. (D) Genotyping PCR showing deletion of Zfp521 allele in genomic DNA extracted from Zfp521hOC−/− long bones cleaned of soft tissues and BM. (E) Von Kossa staining of tibia sections in 3-wk-old global Zfp521−/− mice and Zfp521+/+ littermate controls. (F) Histomorphometric analysis of samples in E (n = 5). (G) Trabecular BV (BV/tissue volume [TV]) at distal femoral metaphysis and in second lumbar vertebra in 3-wk-old Zfp521−/− and control mice measured by μCT (n = 5). (H) Von Kossa staining of tibia sections in 6-wk-old Zfp521hOC−/− mice and littermate controls. (I) Histomorphometric analysis of samples in H (n = 6). (J) Trabecular BV (BV/TV) at distal femoral metaphysis and in second lumbar vertebra in 12-wk-old Zfp521hOC−/− and control mice measured by μCT (n = 5–6). (K) Von Kossa staining of tibia sections in 12-wk-old Zfp521hOC−/− mice and littermate controls. (L) Histomorphometric analysis of samples in K (n = 6). (M) Serum PINP and CTX levels in 3-wk-old global Zfp521−/− mice and Zfp521+/+ littermate controls (n = 6–9). (N) Serum PINP and CTX levels in 6-wk-old Zfp521hOC−/− and control mice (n = 5–6). N.Ob, number of OBs; N.Oc, number of OCs. All data are means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bars: (E) 400 μm; (H and K) 600 μm. See also Tables S1–S3.

Zfp521 favors bone formation in mature OBs. (A) Generation of null and conditional Zfp521 alleles. Zfp521 genomic region encoding exon 4 is shown. Restriction fragment sizes are indicated as well as the positions of the internal and the two flanking probes used for genotyping analysis (Roman numerals). The shaded area indicates the part of the genomic region included in the targeting vector, and the three different alleles are shown. “neo” is the result of the gene-targeting event. “cko” is the conditional knockout allele derived from the neo allele after Flpe-mediated excision of the PGK-neo cassette. One Frt site and two loxP sites remain in the locus. “ko” is the null allele derived from the “neo” or from the “cko” allele by Cre-mediated recombination between the two loxP sites. Only a single loxP site remains in the modified locus. Splicing of exon 3 to exon 5 generates a frameshift. loxP and Frt sites are indicated as closed and open triangles, respectively. Neo, PGK-em7-neomycin dual selection cassette for bacteria and embryonic stem cells; TK, thymidine kinase cassette for counter-selection in embryonic stem cells; X, XbaI; B, BamHI; N, NotI. The genomic region is not drawn to scale. (B) Results of a Southern blot analysis of BamHI-digested tail DNA, probed with the internal probe (III). (C) Northern blot analysis of whole-brain RNA from 3-wk-old mice using a full-length Zfp521 cDNA probe. The blot was rehybridized with a Gapdh probe as a control for RNA quality. wt, wild type; Δexon4, position of the residual mRNA after removal of exon 4. (D) Genotyping PCR showing deletion of Zfp521 allele in genomic DNA extracted from Zfp521hOC−/− long bones cleaned of soft tissues and BM. (E) Von Kossa staining of tibia sections in 3-wk-old global Zfp521−/− mice and Zfp521+/+ littermate controls. (F) Histomorphometric analysis of samples in E (n = 5). (G) Trabecular BV (BV/tissue volume [TV]) at distal femoral metaphysis and in second lumbar vertebra in 3-wk-old Zfp521−/− and control mice measured by μCT (n = 5). (H) Von Kossa staining of tibia sections in 6-wk-old Zfp521hOC−/− mice and littermate controls. (I) Histomorphometric analysis of samples in H (n = 6). (J) Trabecular BV (BV/TV) at distal femoral metaphysis and in second lumbar vertebra in 12-wk-old Zfp521hOC−/− and control mice measured by μCT (n = 5–6). (K) Von Kossa staining of tibia sections in 12-wk-old Zfp521hOC−/− mice and littermate controls. (L) Histomorphometric analysis of samples in K (n = 6). (M) Serum PINP and CTX levels in 3-wk-old global Zfp521−/− mice and Zfp521+/+ littermate controls (n = 6–9). (N) Serum PINP and CTX levels in 6-wk-old Zfp521hOC−/− and control mice (n = 5–6). N.Ob, number of OBs; N.Oc, number of OCs. All data are means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bars: (E) 400 μm; (H and K) 600 μm. See also Tables S1–S3.

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