Complete data set for all the mice comprising the 810 genes screened

Results of the B16-F10 pulmonary metastasis screen for the 810 genes consisting of 13,426 individual mice

Complete listing of the mutant mouse genes screened and the associated allele information

A subset of genes (29) were screened as 2 different alleles and for 1 gene a total of 3 alleles have been screened. The different alleles were generally tm1a and tm1b to discern if there was any difference after cre-mediated excision of the critical exon and targeting cassette (for details of the EUCOMM/KOMP-CSD tm1a/tm1b allele nomenclature refer to Skarnes et al 9). In some instances, one allele was a point mutation and the other targeting the whole gene. ‘Screened gene’ is the gene name as it appears in Data Record 2, followed by the ‘full allele name’, ‘allele superscript’ and ‘allele MGI ID’ (where a record exists). The ‘unique identifier’ is the internal colony prefix that was used for tracking and is specific to the gene and allele within the institute. In some instances, there are multiple colony prefixes for the same gene and allele – this occurred when additional mice were generated for testing outside of the high-throughput screening project. ‘MGI Gene/Marker ID’ allows tracking of the gene independent of any change in gene name for which a number have changed. The ‘current symbol’ is the most recent gene name and ‘synonym screened’ is where the gene has since been changed at MGI (and also internally) so allows for cross checking to the original data. The full current gene name and feature annotation is listed together with the chromosomal location of the gene and the Ensembl ID

Expression of Lrp1b and Lrp4 ECDs.

<p>Whole brain lysates (50 µg) from (<b>A</b>) Lrp1b and (<b>B</b>) Lrp4 truncation mutants were analyzed for expression of the ECD. For the <i>Lrp1b</i> truncation (“Lrp1b EC Stop”), the ECD is expressed at approximately the same size as the full-length receptor (“Wt”) due to the negligible reduction in predicted protein mass. However, as expected the ICD epitope is only present in wild-type tissues. By contrast, in the <i>Lrp4</i> truncation (“Lrp4 EC Stop”), there is a significant shift in size of the ECD protein band compared to full-length receptor. As for Lrp1b, no ICD is detected in the truncated Lrp4 strain. β-Actin was used as a loading control.</p

Blastocyst outgrowth assay.

<p>Time course of <i>Lrp1b</i> wildtype (<i>Lrp1b^+/+</i>) compared to <i>Lrp1b</i> knockout (<i>Lrp1b^{tm1wtsi/tm2wtsi}</i>) trophoblast explant growth, showing expansion of inner cell mass and trophoblast formation. Images were taken on days 1, 2, 4, and 6.</p

Genotyping of Lrp1b heterozygous intercrosses.

<p>Genotyping data at 4 wks, E8.5 and E10.5 was performed on genomic DNA from intercrosses of <i>Lrp1b^tm2wtsi</i> mice, whereas genotyping of the E3.5s was performed on genomic DNA from intercrosses of <i>Lrp1b^tm1wtsi</i> with <i>Lrp1b^tm2wtsi</i> mice. Experimental data was statistically analyzed using Fisher's exact test. NS, not significant.</p

Summary of known mutations and their respective phenotypes.

<p>The known mutations in murine models for (<b>a</b>) <i>Lrp1b</i> and (<b>b</b>) <i>Lrp4</i> are shown. The presence of the extracellular domain (ECD) rescues the lethality caused by the complete functional null mutation.</p

Lrp4 ECD Inhibits Wnt signaling <i>in vitro</i>.

<p>HEK-293 cells were transfected using the TOP-Flash reporter system in the presence of the indicated plasmids (0.5 µg/construct as indicated, where required empty pcDNA3.1 plasmid DNA was added to bring the DNA concentration up to a total amount of 2.5 µg plasmid DNA/condition). Dkk1 and Lrp4 independently inhibit Wnt signaling (lane 5 and 6). Inhibition of Wnt1 induced reporter activation by co-transfection of Lrp4 ECD and Dkk-1 is synergistic (lane 7). Lrp5 and Lrp6 are co-receptors of the frizzled complex and required for Wnt1 mediated activation, however, HEK-293T cells do not express Lrp5 or 6 endogenously and thus need to be co-transfected with the respective plasmids <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009960#pone.0009960-Johnson1" target="_blank">[11]</a>.</p

Generation of <i>Lrp1b</i> null alleles.

<p>(<b>A</b>) Duplication of N-terminal exons 6-8 to generate the <i>Lrp1b^tm1wtsi</i> allele and Southern blot hybridization after <i>EcoR</i>V digestion of embryonic stem cell genomic DNA to verify targeting of the allele. (<b>B</b>) Duplication of C-terminal exon 69 to generate the <i>Lrp1b^tm2wtsi</i> allele and Southern blot hybridization after <i>BamH</i>I digestion of embryonic stem cell genomic DNA to verify targeting of the allele. <i>A</i>, <i>Afl</i>III; <i>B</i>, <i>BamH</i>I; <i>EV</i>, <i>EcoR</i>V; <i>S</i>, <i>Swa</i>I.</p

Lrp4 undergoes regulated intramembraneous processing. (A) Lrp4 ECD release is induced by ADAM10 <i>in vitro</i>.

<p>50 µL of concentrated supernatant and 50 µg of cell lysate were analyzed with a polyclonal antibody detecting either the Lrp4 extracellular domain (Lrp4 ECD) domain (supernatant) or the Lrp4 intracellular domain (cellular extracts). The extracellular domain is present in the supernatant after transfection with Lrp4 and co-transfection with the metalloproteinase Adam10 (lane 4), but not in the absence of Adam10 (lane 3). Immunoblotting for β-actin was used to demonstrate equal loading. <b>(B) Lrp4 ICD is cleaved by γ-secretase.</b> Lrp4 expression in 293T cells reveals bands at 20, 75, and 250 kDa (lanes 2 and 4). The protein levels of 250 and 75 kDa species are independent of DAPT (i.e. γ-secretase inhibitor) treatment. By contrast, the membrane bound ICD at 20 kDa accumulates in the presence of DAPT. No protein products were detected in the untransfected lanes (1 and 3). β-Actin was detected to demonstrate equal loading.</p

Additional file 1: of Somatic drivers of B-ALL in a model of ETV6-RUNX1; Pax5 +/− leukemia

List of targeted exome baits. The file contains a list of genes that were captured as part of the targeted exome sequencing performed as part of this study. (XLSX 53 kb