Figure 1. Generation of Dmp1 null mice by gene targeting. (A) Schematics of mouse Dmp1 wild-type locus with locations of key restriction enzyme sites, and the NLSLacZpA vector (NLS, nuclear localization signal peptide) with the neo cassette inserted into exon 6 at the ApaI and EcoRV sites. Southern blot screening strategy is also displayed. Note that the coding region for 384 out of 503 amino acids, including RGD, has been removed. (B) Representative Southern blot analysis of DNA from a wild-type and a heterozygous ES clone. DNA was digested with BamHI, and hybridized with 5' and 3' probes separately, resulting in a 15-Kb band that corresponds to the wild type. There are two additional BamHI cut sites on the lacZ neo cassette, which gives two bands for the heterozygous ES cell clone: 9.1 Kb for the 5' side and 6.8Kb for the 3' side, respectively. Analysis of the data demonstrates that one of the Dmp1 alleles is replaced by the inserted lacZ cDNA, whose expression will be used to reflect the endogenous Dmp1 expression pattern (see Fig. 2). (C) Northern blot and RT-PCR analyses. The mRNAs were extracted from wild-type (+/+), heterozygous (+/-), and homozygous knock-in (-/-) calvarial cells. PCR amplification was then performed with 2-µL aliquots of the target cDNA. A 5' primer (5'-CGGCTGGTGGACTCTCTAAG-3', corresponding to an oligonucleotide of 374 to 393 of Dmp1 cDNA) and a 3' primer (5'-CGGGGTCGTCGCTCTGCATC-3', corresponding to an oligonucleotide of 750 to 769 of Dmp1 cDNA) were used for Dmp1 RT-PCR. No wild-type Dmp1 mRNA was detected in the homozygous knock-in samples by both Northern and RT-PCR analysis, while a 5.8-Kb fusion transcript containing exons 1-5 of Dmp1 and the lacZ reporter gene was detected in both heterozygous and homozygous knock-in samples. (D) Dmp1 immunostaining. The femurs from neonatal Day 19 wild-type (left) and homozygous knock-in (right) mice were stained by an antibody to DMP1 peptide (105 to 125). There is no detectable signal (brown) in Dmp1 lacZ-knock-in homozygous mice, while a strong signal is obvious in osteocytes in control mice.