<p>(<b>A</b>) DNA analysis of adult mice i.p. injected with vehicle (corn oil) or TM (9 mg/40 g body weight) using the same 3-plex PCR reaction as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015887#pone-0015887-g004" target="_blank">Figures 4A and B</a>. Wild type mice (WT;Cre-) only amplified the wild type allele (lane 1). Doubly transgenic mice (<i>Smn<sup>C-T-Neo/WT</sup>;Cre<sup>Esr1</sup></i>) in the absence of TM (-TM) displayed a low basal level of <i>pgk-neo</i> excision as has been previously reported for this <i>Cre</i> line <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015887#pone.0015887-Hayashi1" target="_blank">[37]</a>. In the absence of the <i>Cre<sup>Esr1</sup></i> transgene, <i>Smn<sup>C-T-Neo/WT</sup></i> mice injected with TM could not excise <i>pgk-neo</i> (lane 3), in all tissues analyzed, <i>pgk-neo</i> excision was only possible and efficient in the presence of <i>Cre<sup>Esr1</sup></i> and TM (lane 4). (<b>B</b>) PCR analysis of E18.5 embryos that received a single i.p. dose of TM (3 mg/40 g body weight) to the pregnant dam at E7.5 or E13.5 DNA was genotyped as above to differentiate <i>Smn<sup>WT</sup></i>, <i>Smn<sup>C-T-Neo</sup></i> and <i>Smn<sup>C-T</sup></i> alleles. Arrows identify the appropriate amplicons. (<b>C</b>) Photomicrograph of E18.5 embryos induced with TM at E7.5 or E13.5. Lines in photograph show where images were tiled together in Photoshop. (<b>D</b>) Western blot and semi-quantitative densitometry of protein extracted from brain tissue of induced and control E18.5 embryos. A small amount of protein was able to be extracted from severely deformed <i>Smn<sup>C-T-Neo/C-T-Neo</sup></i> embryos identified as “escapers” for comparison to induced <i>Smn<sup>C-T-Neo/C-T-Neo</sup>;Cre<sup>Esr1</sup></i> rescued embryos. Semi-quantitative densitometry was performed on a separate blot using the same samples shown and normalized to β-tubulin, without the uninduced mutant. Protein levels from induced homozygous embryos, <i>Smn<sup>C-T-Neo/C-T-Neo</sup>;Cre<sup>ESR1</sup></i>, (0.7±0.10) was greater than <i>Smn<sup>WT/-</sup></i> (0.5±0.2). Abbreviations: (WT) <i>Smn</i> wild type allele, (<i>Cre+</i> and <i>Cre-</i>) presence or absence of <i>Cre<sup>Esr1</sup></i>, (C-T-N/WT) <i>Smn<sup>C-T-Neo/WT</sup></i>, (C-T-N/C-T-N) <i>Smn<sup>C-T-Neo/C-T-Neo</sup></i>, (C-T) <i>Smn<sup>C-T</sup></i> allele, (C-T-Neo) <i>Smn<sup>C-T-Neo</sup></i> allele, (TM) tamoxifen.</p
