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. 2013 Apr 11;288(21):15006–15014. doi: 10.1074/jbc.M113.458448

FIGURE 1.

FIGURE 1.

Generation of mice harboring Dapper3neo-flox or Dapper3 null allele. A, shown is a schematic diagram of the wild-type Dapper3 allele, targeting vector, targeted Dapper3neo-flox, and Dapper3 null allele. Boxes with numbers represent exons. Arrows mark the position of the PCR primers used for genotyping. The location of the 5′ outside and 3′ outside probes for Southern blot analysis are indicated. Solid triangles denote loxP sites; open triangles denote frt sites. Neo and TK are positive and negative selection genes, respectively. Besides restriction site PvuI, two additional sites for Southern blot were introduced in the targeting vector: a BamHI site between 5′ loxP site and Exon 2 and an XbaI site between 3′ loxP site and Exon 4. The 5′ homologous arm of the targeting vector is 5.7 kb, and the 3′ arm is 4.5 kb. Dapper3neo-flox allele is created through homologous recombination between wild-type allele and targeting vector. Dapper3 null allele was conversed from Dapper3neo-flox allele by crossing to transgenic cre mice. B, Southern blot analysis of XbaI digested genome DNA from positive ES cell clones is shown. A 5′ outside probe was used to identify the wild-type (17.3 kb) and targeted allele (9.5 kb) from XbaI-digested genome DNA. The Dapper3+/flox ES cell clones show both 17.3-kb and 9.5-kb bands as expected. wt, wild type; fl, flox. C, PCR genotyping of mice with Dapper3neo-flox allele is shown. The primer set P1F-P1R was used to distinguish the targeted allele (593 bp) from the wild-type allele (497 bp). +/+, wild-type; +/fl, Dapper3+/flox; fl/fl, Dapper3flox/flox. D, generation of mouse embryos harboring Dapper3 null allele is shown. The combination of primer sets P1F-P1R and P1F-P2R could identify three genotypes; only one 497-bp band represents the Dapper3+/+ embryo; only one 434-bp band indicates Dapper3−/− embryo; embryos with both the 497-bp and the 434-bp bands are Dapper3+/−. E, genotyping of MEFs cells by RT-PCR is shown. The primer set pE1-pE4 was used to amplify the wild-type Dapper3 (656 bp) or the truncated Dapper3 (406 bp). Gapdh (100 bp) is used as an internal control. F, immunoblotting (IB) of mouse Dapper3 protein from Dapper3+/+, Dapper3+/−, and Dapper3−/− MEFs cells is shown. The arrow indicates the position of Dapper3. The band below is a none-specific protein.