Genotypes of <i>Smn</i> embryos at E18.5 exposed to tamoxifen at E7.5 or E13.5.

<p>(χ², E7.5 injection p = 0.051; E13.5 injection p = 0.84).</p

Smn induction in adults and embryos from a single injection of tamoxifen.

<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^C-T-Neo/WT;Cre^Esr1</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^Esr1</i> transgene, <i>Smn^C-T-Neo/WT</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^Esr1</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^WT</i>, <i>Smn^C-T-Neo</i> and <i>Smn^C-T</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^{C-T-Neo/C-T-Neo}</i> embryos identified as “escapers” for comparison to induced <i>Smn^{C-T-Neo/C-T-Neo};Cre^Esr1</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^{C-T-Neo/C-T-Neo};Cre^ESR1</i>, (0.7±0.10) was greater than <i>Smn^WT/-</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^Esr1</i>, (C-T-N/WT) <i>Smn^C-T-Neo/WT</i>, (C-T-N/C-T-N) <i>Smn^{C-T-Neo/C-T-Neo}</i>, (C-T) <i>Smn^C-T</i> allele, (C-T-Neo) <i>Smn^C-T-Neo</i> allele, (TM) tamoxifen.</p

Genotype of <i>Smn^C-T-Neo</i> and <i>Smn^2B-Neo</i> intercross progeny.

<p>(+) denotes wild type, (−) denotes either C-T-Neo or 2B-Neo.</p

Generation of mutant <i>Smn</i> alleles.

<p>(<b>A</b>) Gene targeting strategy to introduce the C-T and 2B mutation into <i>Smn</i> exon 7 using the gene targeting vectors pSmnC-T-Neo and pSmn2B-Neo. (<b>B</b>) Southern blot analysis of BamH I and Pst I digested DNA from <i>neo</i> resistant ES cell clones identified homologous recombinants. Two clones from each were used to perform blastocyst injections. (<b>C</b>) Germline transmission of <i>Smn^C-T-Neo</i> and <i>Smn^2B-Neo</i> alleles were determined by direct sequencing of <i>Smn</i> exon 7 PCR products from heterozygous mice. The C-T mutation corresponds to the nucleotide transition within exon 7 of the <i>SMN2</i> gene. The 2B mutation corresponds to a mutation within the splice enhancer region 2B, changing GGA to TTT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015887#pone.0015887-DiDonato2" target="_blank">[32]</a>.</p

Smn expression is efficiently induced from the <i>Smn^C-T-Neo</i> allele <i>in vitro</i>.

<p>Two independent primary MEF cells lines were derived from double transgenic embryos (<i>Smn^C-T-Neo/WT;Cre^Esr1</i>). (<b>A</b>) Schematic of the <i>Smn^C-T-Neo</i> allele from exon 6 to exon 8. Arrows represent forward and reverse primers used in the 3-plex PCR reaction to identify <i>Smn^WT</i> (640 & 637), <i>Smn^C-T-Neo</i> (638 & 637), and <i>Smn^C-T</i> (640 & 637) alleles. Primers 640 and 637 do not amplify a product in the presence of <i>pgk-neo</i> as the amplicon exceeds the time of elongation. (<b>B</b>) 3-plex PCR amplification of DNA from MEF lines 1 and 2 treated for 1 hr with 1 mM tamoxifen (+TM). MEF lines 1 and 2 left untreated (-TM) showed a slight amount of background excision (lanes 2 & 3); however, in the presence of tamoxifen (+TM), they readily amplify the <i>Smn^C-T</i> allele (lanes 4&5). Controls in lanes 6, 7, and 8 were E10.5 embryos harvested to show the indicated genotypes from crosses using germline transmitting <i>Smn^C-T-Neo</i> and <i>Smn^C-T</i> alleles. (<b>C</b>) RNA from untreated (-TM) and induced (+TM) MEF cells were amplified by RT-PCR and <i>FL-Smn</i> transcripts directly sequenced. Induced MEFs (+TM) produced enough <i>FL-Smn</i> transcripts from the mutant C-T allele that could be detected by direct sequencing. The arrow points out the C-T mutation in +TM treated cultures. (<b>D</b>) qRT-PCR of <i>FL-Smn</i> and <i>Δ7Smn</i> from uninduced (-TM) and induced (+TM) cultures. Abbreviations: (TM) tamoxifen (C-T-N/WT;<i>Cre</i>+) <i>Smn^C-T-Neo/+;Cre^Esr1</i> (C-T-N/WT) <i>Smn^C-T-Neo/+</i> (C-T/WT) <i>Smn^C-T/+</i> (C-T-N/C-T) <i>Smn^C-T-Neo/C-T</i>.</p

Whole mount analysis of embryos.

<p><i>Smn^2B-Neo</i> or <i>Smn^C-T-Neo</i> heterozygotes were intercrossed and embryos obtained at either E9.5 or E12.5 for whole-mount analysis and genotyping. (<b><i>a–h</i></b>) E9.5 <i>Smn^C-T-Neo</i> embryos. Heterozygotes (C-T-N/WT) are identical to wild type (WT/WT) littermates. Homozygotes (C-T-N/C-T-N) are small but alive and larger than the <i>Smn^{2B-Neo/2B-Neo}</i> (2B-N/2B-N) homozygotes. (<b><i>i–p</i></b>) E12.5 <i>Smn^C-T-Neo</i> embryos. Homozygotes are extremely small compared to controls and many are being reabsorbed as shown in panel (p). (<b>a'–h'</b>) E9.5 <i>Smn^2B-Neo</i> embryos. Heterozygotes (2B-N/WT) are identical to wild type (WT/WT) littermates. Homozygotes (2B-N/2B-N) are developmentally retarded though still alive with signs of lethality clearly present before this period in some embryos that did not allow for genotyping (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015887#pone-0015887-t001" target="_blank">Table 1</a>). (<b><i>i'–p'</i></b>) E12.5 <i>Smn^2B-Neo</i> embryos. All homozygous mutant embryos are undergoing resorption. Insets in o' and p' are magnified images of embryos in panel. Scale for all E9.5 embryos is 100 µM and for E12.5 200 µM.</p