Targeting strategy to generate a conditional <i>Vps35</i> allele.

<p>(A) LoxP sites were introduced in the third and fourth introns of the <i>Vps35</i> gene by homologous recombination. (B) Southern blot and PCR analysis showing correct targeting of selected ES cell clone. </p

<i>Vps35</i>^{<i>∆/∆</i>} organoids show a growth defect but are competent to respond to Wnt signaling.

<p>(A) Growth was quantified by scoring the organoids in categories based on the number of buds the organoids had produced 5 days after passaging. (B) <i>Vps35</i>^{<i>∆/∆</i>} organoids show reduced proliferation compared to control organoids. This could not be completely rescued by Wnt3a supplemented in the medium (ERN: small intestine organoid medium, containing EGF, R-Spondin, Noggin, WENR: ERN medium supplemented with 30% Wnt3a conditioned medium). (C) <i>Vps35</i>^{<i>∆/∆</i>} organoids can respond to Wnt signaling as assayed by Axin2 qPCR (data are represented as mean ± SD, n=3). (D) Percentage of growing organoids cultured in ENR medium with varying R-spondin concentrations. </p

Knockout of <i>Vps35</i> in intestinal organoids.

<p>Intestinal organoids were obtained from a <i>Vps35</i>^<i>fl/fl</i><i>; Villin-CreERT2</i> mouse and treated with 0.5 μM 4-OHT for 12 hours (<i>Vps35^∆/∆</i>), or control treated (control). (A) PCR analysis of genomic DNA from <i>Vps35</i> knockout organoids shows complete deletion of exon 4 of <i>Vps35 </i><i>in </i><i>vitro</i>. (B) RT-PCR shows absence of <i>Vps35</i> exon 4 from mRNA of <i>Vps35</i>^{<i>∆/∆</i>} organoids. (C) Western blot analysis shows absence of Vps35 protein and reduced Wls protein levels in <i>Vps35</i>^{<i>∆/∆</i>} organoids. (D) <i>Vps35</i>^{<i>∆/∆</i>} organoids show normal morphology, Paneth cells are indicated by arrowheads. (E) RT-PCR analysis of molecular markers of differentiated intestinal cells and intestinal stem cells.</p

Knockout of <i>Vps35 in vivo</i>.

<p>(A) PCR analysis using primers that anneal outside <i>Vps35</i> exon 4 shows deletion of exon 4 from genomic DNA isolated from small intestinal epithelium (SI) of 4-OHT induced <i>Vps35</i>^<i>fl/fl</i> (control) or <i>Vps35</i>^<i>fl/fl</i><i>; Villin-CreERT2</i> mice (<i>Vps35^∆/∆</i>). Histological analysis of <i>Vps35</i> knockout intestine showed no defects in crypt-villus morphology. Intestine sections were Periodic Acid Schiff (PAS) stained (B) and immunohistochemistry was performed to stain Lysozyme (C). </p