Figure 1
From: Rapid generation of endogenously driven transcriptional reporters in cells through CRISPR/Cas9
The development of an endogenously driven, second-generation luciferase and GFP reporter for the TGFβ pathway (2G TGFβ reporter).
a. An outline of the strategy used to generate the 2G TGFβ reporter targeting the PAI-1 gene locus. b. Relative expression levels of PAI-1 mRNA in WT U2OS cells stimulated with TGFβ by qRT-PCR. c. Western blotting of WT U2OS cells stimulated with TGFβ. d. U2OS(SEC-C) control and transfected (sgPAI-1 and the 2G TGFβ reporter donor vector) cells were analysed for GFP expression using FACS. e. Luciferase activity of GFP-expressing single cell clones was measured. f. Distribution of GFP fluorescence intensity in 2G TGFβ reporter cells before and after TGFβ stimulation. g. Wide field fluorescence microscopy imaging of GFP expression in 2G TGFβ reporter cells treated without or with TGFβ. Scale bars represent 20 μm. GFP channel indicated in green and nuclear DAPI staining in blue. h. EtBr-stained agarose gel showing PCR products of genomic DNA samples of U2OS(SEC-C) control and 2G TGFβ reporter (clone-17) cells. PCR products (of Allele-1) were isolated, cloned and sequenced. Clone-17 shows a frame shift insertion as indicated in chromatograms. Bar chart data are represented as mean and error bars indicate standard deviation.
