Genomic configurations at the <i>Ate1</i> locus of <i>Cre-lox</i>-based mouse strains constructed in the present work.

Abstract

<p>(A) The 5′ end of the previously produced unconditional <i>Ate1⁻</i> allele <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007757#pone.0007757-Kwon2" target="_blank">[10]</a>, in which the <i>Ate1</i> exons 1b through 3 were replaced by a cassette encoding a promoter-lacking, NLS-containing LacZ (NLS-βgal) (it was expressed from the endogenous P<i>_Ate1</i> promoter) and the Neo selection marker expressed from the phosphoglycerate kinase P<i>_PGK</i> promoter (green rectangles). (B) A diagram of the 5′ end of wild-type (wt) mouse <i>Ate1</i>, indicating approximate locations of exons 1a through 5. (C) The ∼22.5 kb targeting construct containing a ∼6 kb long-arm region of <i>Ate1</i> homology (shown as a shaded rectangle on the left); a single loxP site (red triangle) upstream of <i>Ate1</i> exon 2, a “floxed”-hygromycin-resistance (<i>hph</i>) cassette, expressed from the P<i>_PGK</i> promoter (blue arrow between two red triangles) downstream of <i>Ate1</i> exon 4; a ∼2 kb short-arm region of homology (an inclined shaded rectangle), and the HSV thymidine kinase (tk) negative-selection cassette expressed from the P<i>_HSV</i> promoter (yellow arrow). Wavy line indicates an abutting sequence of the pBR322 plasmid DNA. (D) The tri-lox <i>Ate1</i> allele obtained after a correctly targeted double crossover event. (E) In the notations here and elsewhere in the paper, “flox-on” indicates a configuration depicted in this panel (the functionally active <i>Ate1^flox</i> allele), whereas “flox-off” indicates a configuration depicted in panel F (the null <i>Ate1⁻</i> allele). The functionally active, “flox-on” (<i>Ate1^flox</i>) allele, obtained by the removal of the <i>hph</i> cassette, using the <i>in vivo</i> expression of Cre-recombinase driven by the P<i>_EIIA</i> promoter, which is active only in pre-implantation blastocysts. (F) The null “flox-off” (<i>Ate1⁻</i>) allele obtained by the inducible expression of CreER recombinase from the P<i>_Cagg</i> promoter and posttranslationally induced by tamoxifen (TM) treatment (see the main text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007757#s4" target="_blank">Materials and Methods</a>). H, approximate locations of HindIII sites used in Southern analyses with DNA probe <b>A</b> (see panel G); E, approximate locations of EcoRI sites used in Southern analyses with DNA probe <b>D</b> (see panel H); black boxes marked “A” and “D” indicate the regions specific for DNA probes <b>A</b> and <b>D</b>, respectively. (G) Southern hybridization analysis using DNA probe <b>A</b> and HindIII-digested genomic DNA. The wt <i>Ate1</i> allele (panel B) yields the 11.8 kb HindIII fragment. The previously constructed <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007757#pone.0007757-Kwon2" target="_blank">[10]</a> unconditionally null <i>Ate1⁻</i> allele (panel A), denoted as “null” on this panel, yields the 9.8 kb HindIII fragment. The functionally active flox-on (<i>Ate1^flox</i>) allele (panel E) yields the 6.3 kb HindIII fragment. Lane 1, <i>Ate1^+/+</i>; lane 2, <i>Ate1^+/−</i>; lane 3, <i>Ate1^+/−</i>; lane 4, <i>Ate1^flox/−</i>. (H) Southern hybridization analysis using DNA probe <b>D</b> (external to targeting vector) and EcoRI-digested genomic DNA. The previously constructed <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007757#pone.0007757-Kwon2" target="_blank">[10]</a> unconditionally null <i>Ate1⁻</i> allele (denoted as “null”) yields the 5.8 kb fragment. Both the wild-type <i>Ate1</i> allele and the flox-on (<i>Ate1^flox</i>) allele yield the 9.7 kB fragment, whereas the null flox-off (<i>Ate1⁻</i>) allele yields the characteristic 3.8 kb fragment. The use of DNA probe <b>D</b> and EcoRI-digested DNA from specific tissues of tamoxifen (TM)-treated <i>Ate1^flox/−</i>;<i>CaggCreER</i> mice allowed approximate estimates of the levels of Cre-mediated recombination that produced the flox-off (<i>Ate1⁻</i>) allele. For example, whereas no flox-on (<i>Ate1^flox</i>) allele could be detected in the kidney and brain of <i>Ate1^flox/−</i>;<i>CaggCreER</i> mice after TM treatment (lanes 5, 6), approximately equal amounts of flox-on (<i>Ate1^flox</i>) and flox-off (<i>Ate1⁻</i>) alleles were present in the heart of TM-treated <i>Ate1^flox/−</i>; <i>CaggCreER</i> mice. Lanes 1–3, 1,000, 250, and 25 ng of EcoRI-digested wt mouse genomic DNA (from a tail biopsy), respectively. Lane 4, EcoRI-digested genomic DNA from the tail of a previously constructed <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007757#pone.0007757-Kwon2" target="_blank">[10] </a><i>Ate1^+/−</i> mouse. Lanes 5–7, EcoRI-digested genomic DNA from the indicated tissues of TM-treated <i>Ate1^flox/−</i>;<i>CaggCreER</i> mice. Lane 8, same as lane 7, but from a TM-treated <i>Ate1^flox/−</i> mouse (lacking the <i>CaggCreER</i> transgene).</p