Establishment of Smap1-targeted mice and SMAP1 expression.
(A) Physical maps of the SMAP1 gene locus and its targeting vector. Features of the recombined and targeted SMAP1 alleles are also depicted. Horizontal lines indicate the genomic sequences. The thick lines indicate the sequence incorporated into the targeting vector. Exon 1, neomycin resistance gene, and diphtheria toxin subunit A gene are indicated. Black and white arrowheads indicate the loxP and frt sequences, respectively. The small rectangle under the line corresponds to the probe that was used for Southern blot hybridization. B indicates a BamHI restriction site. (B) Southern blot analysis of genomic DNA prepared from Smap1+/+ (+/+), Smap1+/– (+/–), and Smap1–/– (–/–) mice. DNA was digested by BamHI and processed for Southern blotting using the hybridization probe shown in A. The wild-type and targeted alleles gave rise to 2.7-kb and 3.3-kb bands, respectively. (C) Immunoblot analysis of protein lysates prepared from bone marrow cells of Smap1+/+, Smap1+/–, and Smap1–/– genotypes. The 50-kDa band represents SMAP1, whereas SMAP2 served as a control. Three independent experiments were performed, and one representative reproducible result is shown. (D) RT-PCR analyses of Smap1 transcripts in bone marrow cells from Smap1+/+ and Smap1–/– mice. Primers were set between exons 1 or 3 and exon 9. HPRT served as a control. (E) SMAP1 expression in hematopoietic cells. Fractions of various hematopoietic lineages were sorted from bone marrow cells of wild-type mice by flow cytometry, and RNA was prepared from each and processed for semiquantitative RT-PCR analyses. DDW, distilled deionized water; -RT, without reverse transcription.