Journal: Cells

Article Title: Generation and Characterization of an Inducible Cx43 Overexpression System in Mouse Embryonic Stem Cells

doi: 10.3390/cells11040694

Figure Lengend Snippet: Inducible Cx43 overexpression in stably transfected, undifferentiated murine G4 ES cells. ( a ) Stably transfected (CAG-floxSTOP-Cx43-P2A-mCherry-Ai6) G4 ES cells of clones 29 and 31 (Cre − ) were transduced with an AAV2.1-Cre virus (Cre + ) to induce transgene expression. ( b ) Transgenic G4 cells prior to AAV2.1-Cre transduction were mCherry − (left picture, shown for C31), and three days after transduction, most of the colonies were mCherry + (middle picture). Single subclones with strong mCherry expression were isolated, and cells were further cultivated (middle and right pictures, shown for C31). Scale bars 100 µm. ( c ) Immunostainings of Cre + G4 ES cells (subclone 31, upper panel) showed, in contrast to Cre − control cells (C31, lower panel), prominent expression of mCherry and of membrane-associated exogenous Cx43 (P2A + ). Total Cx43 (green), P2A-tagged exogenous Cx43 (white), mCherry (red), and Hoechst (blue). Scale bars 10 µm. ( d ) Western blot analysis of Cre − and Cre + cells of C31 proved inducible Cx43 overexpression; note also mCherry − and P2A expression in the AAV2.1-Cre treated cells. * p value ≤ 0.05.

Article Snippet: The customized 1968 bp DNA fragment was cut out by digestion with FseI, isolated and ligated downstream of the CAG promoter and a floxed stop cassette into the FseI sites of the Ai6 vector (Addgene, Watertown, MA, USA, #22798) [ ], thereby exchanging the zsGreen reporter.

Techniques: Over Expression, Stable Transfection, Transfection, Clone Assay, Transduction, Expressing, Transgenic Assay, Isolation, Western Blot

Journal: Cells

Article Title: Generation and Characterization of an Inducible Cx43 Overexpression System in Mouse Embryonic Stem Cells

doi: 10.3390/cells11040694

Figure Lengend Snippet: Inducible Cx43 expression and functional gap junction formation in stably transfected HeLa cells. ( a ) Scheme of single cell dilution of stably transfected (CAG-floxSTOP-Cx43-P2A-mCherry-Ai6, Cre − ) and transduced (AAV2.1-Cre, Cre + ) HeLa cells. Scale bars 100 µm. ( b ) Immunostainings of transgenic HeLa cells confirmed strong expression of exogenous Cx43 and P2A in the plasma membrane of mCherry + cells (Cre + , upper panel), whereas not-transduced control cells were Cx43 − , P2A − , and mCherry − (Cre − , lower panel). Scale bars 10 µm. ( c ) Western blot analysis confirmed expression of Cx43, P2A, and mCherry in Cre + HeLa cells, whereas none of these proteins were detected in Cre − and WT HeLa cells. ( d ) Single Cre + or Cre − HeLa cells were dialyzed via a patch pipette with the small MW dye Alexa350 (349 Da), and dye passage to closely adjacent cells was observed in Cre + cells (upper panel; n = 4). Cre − HeLa control cells did not show passage of Alexa 350 to adjacent cells (lower panel, n = 5). Dialyzed cell marked by a yellow circle; after dye diffusion positive cell colonies marked by a yellow dotted line. Scale bars 100 µm.

Article Snippet: The customized 1968 bp DNA fragment was cut out by digestion with FseI, isolated and ligated downstream of the CAG promoter and a floxed stop cassette into the FseI sites of the Ai6 vector (Addgene, Watertown, MA, USA, #22798) [ ], thereby exchanging the zsGreen reporter.

Techniques: Expressing, Functional Assay, Stable Transfection, Transfection, Transgenic Assay, Western Blot, Transferring, Diffusion-based Assay

Journal: Cells

Article Title: Generation and Characterization of an Inducible Cx43 Overexpression System in Mouse Embryonic Stem Cells

doi: 10.3390/cells11040694

Figure Lengend Snippet: Inducible overexpression of functional Cx43 gap junctions in stably transfected 3T3 cells. ( a ) Immunostaining of stably transfected (CAG-floxSTOP-Cx43-P2A-mCherry-Ai6, Cre − ) and transduced (AAV2.1-Cre, Cre + ) mCherry + 3T3-fibroblasts showed strong Cx43 and P2A expression (upper panel). In contrast, Cre − 3T3 cells exhibited much lower Cx43 expression and expressed neither mCherry fluorescence nor P2A. Upper and lower left pictures: Hoechst (blue) and P2A (white); insets: Hoechst (blue), Cx43 (green), mCherry (red), and P2A (white). Scale bars 20 µm. ( b ) Western blot analysis of transgenic Cre + cells proved Cx43 and mCherry (over-)expression but not in control cells. Quantification of Cx43 expression proved its strong overexpression in Cre + 3T3 cells. ( c ) Fluorescence recovery after photobleaching (FRAP) measurements illustrated significantly faster calcein AM dye recovery in transgenic Cre + 3T3-fibroblasts (clone 2) than in either WT or Cre − 3T3-fibroblasts (clone 2); FRAP recovery rates were quantified at 4 and 10 min after bleaching (quantification times are marked by yellow arrows). ( d ) Original fluorescence pictures of 3T3 Cre + , Cre − , and WT fibroblasts, respectively, after calcein AM (0.38 µM) dye loading; bleaching (for 5 s with a 561 nm laser with 2.5 mW intensity) of a single fibroblast (bleached cell marked by a yellow dotted line and a yellow arrow); and dye recovery at 17 s, 37 s, and 57 s after bleaching; calcein AM dye (green). Scale bars 20 µm. ** p value ≤ 0.01, *** p value ≤ 0.001, **** p value ≤ 0.0001, ns (not significant) p value > 0.05.

Article Snippet: The customized 1968 bp DNA fragment was cut out by digestion with FseI, isolated and ligated downstream of the CAG promoter and a floxed stop cassette into the FseI sites of the Ai6 vector (Addgene, Watertown, MA, USA, #22798) [ ], thereby exchanging the zsGreen reporter.

Techniques: Over Expression, Functional Assay, Stable Transfection, Transfection, Immunostaining, Expressing, Fluorescence, Western Blot, Transgenic Assay