Genotype analysis from timed-pregnant matings of <i>Snap23</i>^Δ/wt heterozygote mice.

<p>Embryo age, number of dissected embryos, and <i>Snap23</i> genotype results are summarized as indicated.</p

<i>Snap23</i>^Δ/Δ blastocysts die prior to uterine implantation.

<p>(<b>A</b>) To evaluate the timing of embryonic lethality, embryos were collected from super-ovulated <i>Snap23</i>^Δ/wt females mated with <i>Snap23</i>^Δ/wt male mice by uterine flushing at E3.5. About 1/4 of the isolated blastocysts were morphologically abnormal and appeared to be degenerating; unlike sibling normal blastocysts they failed to develop any further during 24 hrs of culture (indicated by red arrows; see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018444#pone-0018444-t002" target="_blank">Table 2</a>). (<b>B</b>) Representative example of genotyping analysis revealing that abnormal blastocysts are homozygous for the <i>Snap23</i> deleted allele (<i>Snap23</i>^Δ/Δ). Genomic DNA was isolated from individual blastocysts (shown in panel (A)) following 24 hr in culture, and genotyping was conducted using primers genoE2 SS, genoE2 AS, and genoE3 rev. PCR products for the <i>Snap23</i>^wt allele (266 bp) and for the <i>Snap23</i>^Δ allele (492 bp) are indicated.</p

Oligonucleotide primers used in this study and estimated size of PCR products.

<p>The oligonucleotide sequences used in this study are listed in the upper table. The oligonucleotide primers were used to genotype the mice, to obtain genomic fragments of <i>Snap23</i>, and to generate probes for Southern blot analysis. The estimated sizes of PCR products obtained during genotyping the mice are indicated in the lower table.</p

Expression of SNAP-23 protein is reduced by half in <i>Snap23</i>^fl/wt and <i>Snap23</i>^Δ/wt mice.

<p>(<b>A</b>) <i>Snap23</i>^fl/wt heterozygous mice were mated and twelve two-week old pups from this mating were genotyped and analyzed for SNAP-23 protein expression. Genotyping of tail DNA was performed using PCR Primer set 2 from tail DNA. A single small PCR fragment (266 bp) is present in <i>Snap23</i>^wt/wt pups, and double fragments (266 and 400 bp) is present in <i>Snap23</i>^fl/wt pups. No homozygous <i>Snap23</i>^fl/fl pups were obtained when more than 50 pups were analyzed from <i>Snap23</i>^fl/wt heterozygous matings. For immunoblot analysis, whole brain was solubilized in modified RIPA lysis buffer and protein levels were analyzed by immunoblotting (WB) as indicated antibodies. (<b>B</b>) <i>Snap23</i>^Δ/wt heterozygous mice were mated and pups from this mating were genotyped and their brains were analyzed for expression of SNAP-23 and other SNARE proteins. Genotyping of tail DNA was performed using PCR Primer set 2. A single small PCR fragment (266 bp) is present in <i>Snap23</i>^wt/wt pups, and double fragments (266 and 492 bp) is present in <i>Snap23</i>^Δ/wt pups. No homozygous <i>Snap23</i>^Δ/Δ pups were ever obtained from <i>Snap23</i>^Δ/wt heterozygous matings. Whole brains were solubilized and analyzed by immunoblotting (WB) using the indicated antibodies.</p