Doxycycline Responsive Transactivator Tet On 3g, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Expandable Arterial Endothelial Precursors from Human CD34+ Cells Differ in Their Proclivity to Undergo an Endothelial-to-Mesenchymal Transition"
Article Title: Expandable Arterial Endothelial Precursors from Human CD34+ Cells Differ in Their Proclivity to Undergo an Endothelial-to-Mesenchymal Transition
Journal: Stem Cell Reports
Figure Legend Snippet: MYCN and SOX17 Induce and Expand Endothelial Precursors (A and B) Experimental approach. (A) Vectors used. Vectors were integrated in cells by the PiggyBac transposase. The promoter EF1α drives constitutive expression of Tet-On 3G . In the presence of doxycycline, Tet-On 3G binds the TRE3G promoter, upregulating expression of MYCN and SOX17 (encoded on separate vectors). (B) The three vectors from (A) were introduced by electroporation into human CD34 + cells (cultured for two days prior to electroporation). The electroporated cells were then cultured in the presence of doxycycline to induce colony formation and expansion. (C) Example colonies arising 9 days after electroporation as described in (B). Phase-contrast images. Scale bars, 400 μm. (D) Efficiencies of colony formation after 9 days. The boxes indicate the maximum to minimum efficiencies from at least two independent experiments; the horizontal lines within the boxes indicate the means. CB, cord blood; ABM, adult bone marrow. The range of the ages of the adult bone marrow donors is provided in years. (E) Growth curve, results are the average ± SD from six independent cell lines, three derived from cord blood and three derived from adult bone marrow. (F) Example phase-contrast images of endothelial precursor cell lines. Scale bars, 400 μm. (G and H) Cell lines were maintained in culture by the ectopic expression of MYCN and SOX17 (100 ng/mL doxycycline) and matured by downregulating the factors for 4 days (10 or 0 ng/mL doxycycline). 293T cells served as negative controls. Results are from two independent cell lines, one derived from cord blood and the other derived from adult bone marrow. (G) Analysis of the endothelial markers CDH5 and PECAM1 by flow cytometry. The number of days indicates the time in culture from the induction of the ectopic expression of MYCN and SOX17 . (H) Analysis of the uptake of fluorescently labeled acetylated low-density lipoprotein (Ac-LDL) by flow cytometry. .
Techniques Used: Expressing, Electroporation, Cell Culture, Derivative Assay, Flow Cytometry, Cytometry, Labeling