Off-target analysis of ZFN gene edited <i>Dkk3b</i>^<i>CFP</i> mouse (Founder #19).

<p>Mouse C57bl6 genome GRCm38; mismatched bases are italicized and underlined.</p

Characterization of the DKK3b:ß-TrCP:ß-catenin complex and its effects of ß-catenin nuclear translocation/signaling.

<p>(A) Co-IP of DKK3b, ß-TrCP and ß-^S33Ycatenin from HEK293 cell lysates. Epitope tagged targets were expressed by transient transfection in HEK293 cells, immune precipitates collected by Protein A/G Sepharose, and co-precipitating partners were analyzed by immunoblot with epitope specific antibodies. (B) Co-IP of HEK293 cell lysates lacking one binding partner. (C) shRNA knockdown of ß-TrCP in HeLa cells. Immunoblots done with anti-TrCP IgG. (D) Effects of <i>ß-TrCP</i> KD and ß-TrCP rescue on TAT-DKK3b dependent inhibition of TOPflash activity in HeLa cells. Unaltered, non-silencing control, <i>ß-TrCP</i> KD cells, and rescued <i>ß-TrCP</i> KD cells expressing a mouse ß-TrCP rescue plasmid were stimulated with ±LiCl for 16h in the absence or presence of TAT-DKK3b (5 μg/ml). TOPflash activity reported as fold change from resting HeLa cells. Data are reported as the means ± se (n = 4); each experiment was repeated 3 times.</p

Schematic diagram of the novel regulatory role of DKK3b in the Wnt/ß-catenin signaling pathway.

<p>DSH, Disheveled; GSK-3, Glycogen synthase kinase 3 beta; CK1, Casein kinase 1; PP2A, Protein Phosphatase 2A; APC, Adenomatous Polyposis Coli; ß-TrCP, ß-Transducin Repeat-Containing Protein; Ub, ubiquitin.</p

Correction: The <i>Dkk3</i> gene encodes a vital intracellular regulator of cell proliferation

Correction: The <i>Dkk3</i> gene encodes a vital intracellular regulator of cell proliferatio

Analysis of the biological role the TSS2 driven <i>Dkk3b</i> in the ZFN gene-edited <i>Dkk3b</i>^<i>CFP/+</i> mouse.

<p>(A) Inhibition of DNA methyltransferase activity increases TSS2-driven CFP in <i>Dkk3b</i>^<i>CFP/+</i> cells. (B) DKK3 isoforms present in wild type and <i>Dkk3b</i>^{<i>CFP/mCherry</i>} MEFs (anti-DKK3 (ab186409) (Abcam). (C) QPCR analysis of <i>Dkk3</i> and <i>Dkk3b</i> transcripts in wild type and <i>Dkk3b</i>^{<i>CFP/mCherry</i>} MEFs. Data reported as means ± se, n = 3. (D) TSS2-driven, immunoreactive CFP expression in representative tissues of the <i>Dkk3b</i>^{<i>CFP/wt</i>} mouse. NSB, Normal rabbit serum, CFP, anti-CFP IgG(cat#632381) (TakaRa).</p

Outcome of crosses of the <i>Dkk3b</i>^{<i>CFP/wt</i>}, Cre-rescued <i>DKK3b</i>^{<i>ΔCFP/wt</i>} mutant and <i>Dkk3b</i>^<i>wt/wt</i> mice.

<p>Outcome of crosses of the <i>Dkk3b</i>^{<i>CFP/wt</i>}, Cre-rescued <i>DKK3b</i>^{<i>ΔCFP/wt</i>} mutant and <i>Dkk3b</i>^<i>wt/wt</i> mice.</p

Effects of TAT-DKK3b on the dynamics of ß-catenin nuclear translocation.

<p>(A) Representative photomicrographs of the distribution of ß-catenin in control (unstimulated) and LiCl-stimulated HeLa cells ± TAT-DKK3b (arrows—cell nucleus). (B) Time course of LiCl-induced nuclear translocation of ß-catenin in HeLa ± TAT-DKK3b (5 μg/ml). At least 200 cells were counted in 20 random fields from 3 individual slides; data reported as means ± SD. (C) Time course of the effects of TAT-DKK3b on steady-state nuclear ß-catenin in LiCl-stimulated HeLa cells. Nuclear ß-catenin positive cells were scored as in B, and the data reported as means ± SD of 3 independent experiments. (D) Immunoblot of the effects of TAT-DKK3b on ß-catenin levels in HeLa cell lysates. Data reported as means ± se, n = 3. (E) Time dependent assembly of the TAT-DKK3b:ß-TrCP:ß-catenin complex in LiCl-stimulated HeLa cells. Cell lysates were incubated with anti-DKK3b-IgG or NRS-conjugated DynaBeads, and co-precipitating proteins determined by immunoblot with anti-target antibodies.</p

Characterization of DKK3b loss of function in MEFs.

<p>(A) QPCR analysis of shRNA knockdown of <i>Dkk3</i> and <i>ß-catenin</i> in MEFs. Data reported as means ± se, n = 3. (B) Basal TOPflash activity in shRNA KD MEFs. Data reported as the means ± se, n = 6. (C) Quantification of unattached, viable cells after 72 h in <i>Dkk3/Dkk3b</i> KD MEFs. Viability determined by Trypan blue exclusion; data reported as means ± se, n = 3. (D) Representative photomicrographs of GFP-expressing, shRNA KD MEFs. TAT-DKK3b (1 μg/ml) was added to the growth medium as indicated. GFP positive KD cells in 10 random fields were counted for each KD condition and the data reported as the means ± SD. (E) Quantitative analysis of the attached cells in part D. A total of 100 cells counted and the data expressed as cells/mm². Data represent the means ± se, n = 6 wells for each condition. Taken together, these data show that our promoter-trap knock-in gene-editing strategy: i) selectively eliminated expression of the intracellular DKK3b; and ii) preserved expression of the secreted DKK3. Loss of DKK3b in MEFs led to elevated nuclear ß-catenin levels, increased ß-catenin signaling, and defective cell attachment to the substratum.</p

Identification of multiple transcripts originating for the <i>Dkk3</i> gene locus.

<p>(A) Schematic diagram of the <i>Dkk3</i> gene (NC_000073.6) in the wild type and <i>Dkk3</i>^{<i>tm1Cni</i>} mouse. Initiator methionine’s for <i>Dkk3</i> (NM_0154814) and <i>D2p29</i> (AF245040) indicated by arrows; CpG islands indicated by red box; LacZ-pA stop cassette in yellow. (B) Quantitative PCR of <i>Dkk3</i> containing exon 2 and exon 3 transcripts in total brain RNA from the <i>Dkk3</i>^{<i>tm1Cni</i>} mouse. Arrows indicate PCR primer sites. Data reported as means ± se of 3 individuals; each sample determined in quadruplicate. (C) Schematic diagram of rat <i>Dkk3</i> intron 2-luciferase reporter constructs. Arrows indicate orientation and location of intron 2 segments upstream of exon 3; data reported as means ± se of 3 independent experiments, each sample determined in triplicate. (D) ChIP analysis of RNA pol II and TATA box binding protein (TBP) binding to the ~66 nucleotides (nt6682-nt6948) of intron 2 adjacent to exon 3 in the rat astrocyte <i>Dkk3</i> gene; data reported as means ± se of 3 independent experiments; each sample determined in quadruplicate.</p

Targeting strategy for ZFN gene-editing of the <i>Dkk3b</i> locus in the mouse.

<p>(A) Organization of exons 2–4 of the wild type <i>Dkk3</i> locus. TSS1, transcriptional start site 1; TSS2, transcriptional start site 2 and ZFN targeting site. (B) Organization of the HR donor. C. Schematic diagram of the gene edited <i>Dkk3b</i>^<i>CFP</i> locus. Target locus modification confirmed using PCR primers anchored outside of the HR region (LF and RR) and overlapping in the CFP cds (LR and RF). PCR products (LF:LR and RF:RR) were sequenced in both directions. Genotyping PCR primers indicated by arrows (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181724#pone.0181724.t001" target="_blank">Table 1</a> for sequences).</p