Clustering of changed genes around <i>SAP102/dlgh3</i> and <i>PSD-95/dlgh4</i> loci. <i>A</i>, significant genes (x-axis) from SAP102^−/Y hippocampus along chromosome X (y-axis), indicating the location of SAP102 gene (<i>dlgh3</i>) and <i>hprt.</i>

<p>White histogram, results from 129S5 strain animals; black histogram, results from C57BL/6j after selection of <i>hprt</i> deletion (see text). <i>B</i>, probe sets from PSD-95^−/− along chromosome 11, where the position of the mutated gene (<i>dlgh4</i>) is indicated. White histogram, hippocampus mRNA; black histogram; cortex mRNA.</p

PCR genotyping results for Atp7a and Dlgh3 genomic regions.

<p>Two bands were observed for Atp7a reactions, one of 694 bp corresponding to 129-derived sequences and another of 637 bp corresponding to C57Bl6-derived sequences (upper panel). The appearance of the 694 bp-band coincided with the presence of the SAP102 mutation (-/y) (lower panel). Wild-type 129S5 (129) and C57BL/6j (BL6) were used as positive controls.</p

Distribution of significant genes between all the chromosomes.

<p>χ² tests were performed in the available mutants under the null hypothesis of random distribution: Random, equal proportion; Norm.1, proportion normalized by chromosome length; Norm.2, normalized by number of genes in each chromosome (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001303#s4" target="_blank">Materials and Methods</a>): *, p<0.1; **, p<0.05; ***, p<0.01; ****, p<0.001. Percentages are the contribution of the chromosome for each ablated gene to the deviation from each random distribution and were calculated as proportion of the χ² values. If a chromosome other than that encoding the knock-out gene had more contribution is stated in “Other chrom”. Total genes are the number of the retrieved significant genes.</p

List of significant gene expression probe sets from X chromosome in SAP102^−/Y samples.

<p>Affy IDs in bold are probe sets that appeared in 129S5 and C57BL/6 <i>hprt</i> wild-type; Affy IDs in italic are probe sets only appearing in C57BL/6 <i>hprt</i> wild-type. The difference in probe sets were likely due to differences in the statistical power of both analyses (ten 129S5 samples <i>vs.</i> seven C57BL/6 samples). When two numbers appear in the “Fold change” column, the upper number is the fold change obtained from 129S5 samples and the lower number from C57BL/6 samples. Chromosomal location denotes the first nucleotide position of the probe set (Ensembl release 45).</p

Validation of microarray analyses by qRT-PCR assays in PSD-95 and SAP102 mutants.

<p>Hippocampus mRNA was extracted and level of mRNA detected using microarrays or qRT-PCR. <i>A</i>, SAP102 mutant mice; results for SAP102, Hprt, Fundc2, BB315069 (3′ to Fundc2) are shown. B. PSD-95 mutant mice; results for PSD-95, Fbxo39 and BG092359 (3′ to Fbxo39). y-axis: fold change of mutant mRNA levels compared to control; mean±s.e.m. P, qRT-PCR data; M, microarray data. M1, M2… refers to different probe sets for the same gene. Note different scale of y-axes.</p

Distribution of significant genes within the chromosome of the ablated gene.

<p>χ² tests were performed in the available mutants under the null hypothesis of random distribution (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001303#s4" target="_blank">Materials and Methods</a>): **, p<0.05; ***, p<0.01; ****, p<0.001. <i>P</i>-values were calculated comparing the region containing the significant genes with the rest of the chromosome (Student's t-tests). ^†, when the clustering did not allow the test or the test failed, the most significant region was calculated using different sizes of genomic region by trial-and-error. Region of significance is expressed in Mb and the correspondent cM. The % of genes contained therein is also expressed.</p

Number of significantly changed genes on targeted chromosome for Foxa3, Neb, Ercc1 and Fibulin-4 mutants.

<p>y-axis: number of changed genes, x-axis: chromosomal position. The arrow indicates the location of the mutated genes in each case.</p

List of significant gene expression probe sets on chromosome 11 from PSD-95^−/− mutants.

<p>Affy IDs in bold are probe sets that appeared in hippocampus and cortex; Affy IDs in italic are probe sets only appearing in cortex. The difference in probe sets within both tissues was likely due to differences in the statistical power of both analysis (9 hippocampal samples <i>vs.</i> 4 cortex samples). When two numbers appear in the “Fold change” column, the upper number is the fold change obtained from hippocampus and the lower number from cortex. Chromosomal location denotes the first nucleotide position of the probe set (Ensembl release 45).</p

Expression level can be increased when selectable marker is removed.

<div><p>(<b>A</b>) Diagram of orientation of constructs in cell lines.</p> <p>(<b>B</b>) A2B2 and A2B2<i>Δ</i> cells induced with doxycycline (<b>i</b>) A2B2 phase contrast (<b>ii</b>) A2B2 EGFP expression (<b>iii</b>) A2B2<i>Δ</i> phase contrast (<b>iv</b>) A2B2<i>Δ</i> EGFP expression</p> <p>(<b>C</b>) Graph of expression levels in induced and uninduced cell lines quantitated by fluorimetry.</p> <p>Expression of EGFP is higher when these cell lines have lost the selectable marker cassette (<i>F</i>(1,40) = 77.25, <i>p</i><0.0001).</p> <p>The orientation of the TRE EGFP makes no difference to expression level (<i>F</i>(1,40) = 1.14, <i>p</i> = 0.2910).</p> <p>Each point is an average of three measurements each from two independently targeted cell lines.</p> <p>Error bars denote standard deviations. Asterisks indicate statistically significant differences.</p></div

Targeting strategy for introducing to ROSA26locus.

<div><p>(<b>A</b>). Diagram of targeting constructs to introduce Dox responsive transgenes into locus.</p> <p>A1 and A2 introduce the same activator transgene in opposite orientations.</p> <p>B1 and B2 introduce the same responder transgene in opposite orientations.</p> <p>(<b>B</b>) Four different cell lines were produced with the activator and responder transgenes in different orientations (<b>C</b>) Southern blots probed with 5′ and 3′ flanking probes produce the correct band sizes to demonstrate appropriate targeting of constructs.</p></div