The <i>Mesd</i> conditional allele.

<p>(<b>A</b>) Comparison of the <i>Mesd Wild-type, Floxed,</i> and <i>LoxP Mesd</i> alleles and targeting vector. The <i>Wild-type Mesd</i> allele (top map) has three exons (1–3) that are designated by grey rectangles. The first exon encodes the signal peptide that directs the MESD protein into the ER as well as the N-terminal helical region essential for maturation of LRPs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075782#pone.0075782-Hsieh1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075782#pone.0075782-Kohler1" target="_blank">[3]</a>. The 3′ untranslated portion of the third exon is indicated by light grey. In the <i>Mesd</i> targeting vector, we introduced the <i>Neomycin resistance</i> gene (<i>Neo</i>) (white rectangle) flanked by Flp-recombinase sites (green triangles) into the <i>Mesd</i> first intron. In addition, we introduced three LoxP sites (red triangles) flanking the first and second exons and the <i>Neo</i> gene.The targeting construct was introduced into C57BL/6 embryonic stem cells (<i>Ozgene</i>), and cell lines heterozygous for the <i>Floxed</i> allele (middle map) were identified by Southern analysis using the 5P, enP, and P3 probes, whose positions are indicated by black rectangles (described in Materials and Methods and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075782#pone-0075782-t001" target="_blank">Table 1</a>). Based on Southern analysis using these probes, we determined that only the first two LoxP sites were incorporated into ES cells; the recombination sites are indicated by dashed lines. Expression of Cre-recombinase will trigger recombination between LoxP sites to generate the <i>LoxP</i> allele (bottom map). (<b>B</b>) Heterozygous <i>Floxed</i> mice were mated to C57BL/6 mice (left) for strain propagation or to animals homozygous for a ubiquitously expressed Cre-recombinase (right) to generate the recombined <i>LoxP</i> (knockout) allele. Progeny were genotyped by Southern analysis of <i>BamH</i>I digested DNA using the enP probe. This probe clearly distinguished the <i>Wt</i> (20 kb) fragment from the <i>Floxed</i> (7.4 kb) or <i>LoxP</i> (4 kb) fragments.