irfp670 construct  (VectorBuilder GmbH)

Journal: eLife

Article Title: On demand expression control of endogenous genes with DExCon, DExogron and LUXon reveals differential dynamics of Rab11 family members

doi: 10.7554/eLife.76651

Figure Lengend Snippet: ( A ) Spinning disk confocal image of A2780 cells with antiGFPnanobody-mCherry knock-in to Rab11a locus stably expressing LifeAct-mtagBFP (gray). Signal of mCherry (red; =mCH) reports full-length integration only of mCherry, not of antiGFPnanobody (scale bar=20 μm). ( B ) Immunoblots of wild-type (wt) A2780 control (CTRL), antiGFPnanobody-mCherry knock-in to Rab11a locus, or A2780 overexpressing stably integrated full-length antiGFPnanobody-mCherry-Rab11a (lentiviral transduction, expected protein size 69 kDa marked by blue arrow). Blots were probed with anti-mCherry (RFP), anti-Rab11a, or anti-Rab11 (targeting both Rab11a/b) antibodies (a=Rab11a; b=Rab11b). Tubulin (Tub), loading control. ( C ) Chromatogram of mCherry-antiGFPnanobody-Rab11a cells (classical knock-in) with schematic of knock-in outcome (seq. prim=sequencing primer). Graph, made by TIDER analysis, estimates the frequency of mutations/indels in knocked-in cells .

Article Snippet: Transfected construct (synthetic) , pLenti Lifeact-iRFP670 BlastR , DOI: 10.1038/s41467-018-05367-2 , RRID: Addgene_84385 , Lentiviral construct to transfect and express LifeAct..

Techniques: Knock-In, Stable Transfection, Expressing, Western Blot, Control, Transduction, Sequencing

Journal: eLife

Article Title: On demand expression control of endogenous genes with DExCon, DExogron and LUXon reveals differential dynamics of Rab11 family members

doi: 10.7554/eLife.76651

Figure Lengend Snippet: ( A ) Immunoblots of A2780 cells lysates probed with antibodies specific for anti-Rab11a, targeting both Rab11a/b (Rab11) or Rab25 (a=Rab11a; b=Rab11b). Tubulin, loading control. ( B ) Schematic of the DExCon-mNeonGreen knock-in strategy and lentiviral transduction for doxycycline (dox)-dependent re-activation of Rab25 expression. ( C–D ) Rab25 DExCon knock-in cells were dox treated for 24–72 hr, sorted for mNeonGreen fluorescence followed by cell growth 2 weeks without dox before re-analysis. Live fluorescence images of re-activated Rab25 fused to mNeonGreen (mNG) in A2780 24 hr after dox treatment (250 ng/ml). ( C ) Top: Cells on tissue culture-treated plastic (scale bar=100 µm). Bottom: Cell in cell-derived matrix (CDM) (spinning disc confocal image; scale bar=20 µm). ( D ) Cells exposed to increasing dox concentration imaged by brightfield and fluorescence microscopy (mT.B=Teton3G-T2A-mTagBFP; brig=brightfield; Ctrl=unmodified cells). Scale bar=100 μm. ( E ) Immunoblots of mNeonGreen-Rab25 DExCon re-sorted cells as indicated in and re-induced with dox (200 ng/ml). Lysates were probed with antibodies specific for Rab25 or Tubulin. Black arrow indicates endogenous Rab25 in OVCAR-3 cells. ( F ) Spinning disc confocal images of mNeonGreen-Rab25 DExCon cells (Rab25, green; Teton3G, blue), re-sorted as indicated in . Scale bar=100 μm. ( G ) Schematic illustration of the photoactivatable split PA-Tet-OFF and PA-Tet-ON constructs combined with DExCon (LUXon). ( H ) Spinning disc confocal images (20 × [left] or 63 × [right] objectives) of mNeonGreen-Rab25 LUXon cells (Rab25 in green) expressing PA-Tet-OFF with mCherry-NLS reporter (red) 18 hr after being illuminated by blue light (10 hr) with or without dox treatment. Orthogonaere imaged live while recycll views of top (1) or two bottom cells (2) are also shown with kymograph corresponding to white dashed line (1 s interval, total 1 min). Scale bar: 10 μm. See – . ( I ) Schematic of 3D cell-zone exclusion invasion assay; 1–4 indicate zones with different light illumination intensities across same well, and these zones are also indicated in ( J ): mNeonGreen-Rab25 LUXon cells (Rab25 in blue) expressing PA-Tet-OFF with mCherry-NLS as nuclear reporter (red) migrated for 24 hr into cell-free collagen matrix labeled by fibronectin (FN-647 shown as green) while being illuminated by blue light of varying intensity. Dashed line indicates scratch and white arrows indicate the most invasive cells. Scale bar=100 µm. See . ( K ) Spinning disc confocal images of spheroids formed by mNeonGreen-Rab25 LUXon (PA-Tet-OFF2) cells illuminated with blue light for different times across 2 days as indicated (total 4 hr vs 21 hr). Cells invading collagen matrix supplemented with FN-647 (magenta) were labeled with Hoechst 3342 (blue) for 1 hr prior imaging. Merge of all three channels, white rectangles for the mNeonGreen (Rab25, green) channel only, or zoom of the yellow rectangle is shown. Scale bar=100 µm. ( L ) Schematic of spheroid invasion assay ‘on chip’ and illumination protocol used in ( M ). mNeonGreen-Rab25 LUXon (PA-Tet-OFF2) cells (scale bar=1 mm) together with ( N ) quantification (n=20–32 from three independent experiments, one-way ANOVA Tukey post hoc test). All schematic illustrations were created with BioRender.com .

Article Snippet: Transfected construct (synthetic) , pLenti Lifeact-iRFP670 BlastR , DOI: 10.1038/s41467-018-05367-2 , RRID: Addgene_84385 , Lentiviral construct to transfect and express LifeAct..

Techniques: Western Blot, Control, Knock-In, Transduction, Activation Assay, Expressing, Fluorescence, Derivative Assay, Concentration Assay, Microscopy, Construct, Invasion Assay, Labeling, Imaging

Journal: eLife

Article Title: On demand expression control of endogenous genes with DExCon, DExogron and LUXon reveals differential dynamics of Rab11 family members

doi: 10.7554/eLife.76651

Figure Lengend Snippet:

Article Snippet: Transfected construct (synthetic) , pLenti Lifeact-iRFP670 BlastR , DOI: 10.1038/s41467-018-05367-2 , RRID: Addgene_84385 , Lentiviral construct to transfect and express LifeAct..

Techniques: Modification, Transfection, Construct, shRNA, Recombinant, Expressing, Plasmid Preparation, CRISPR, Knock-In, Labeling, Sequencing, Software, Invasion Assay