Figure 2.

Generation of βTC3 G6pc2 knockout and control cell lines using CRISPR-Cas9.A, schematic providing an overview of βTC3 G6pc2 KO cell line development using CRISPR-Cas9 (See Experimental procedures for details). B, mKate2 and brightfield superimposed images from three biological replicates of βTC3 cells expressing Cas9 protein. The single cell clone with the highest mKate2 fluorescence, shown here, was selected for CRISPR RNA transfection. C, TIDE analysis showing the total percentage of insertions and deletions (indels) around three different loci targeted with three different CRISPR RNA sequences in four biological isolates of the single cell clone shown in panel (B). D, verification of loss of G6pc2 functional activity in KO cells by measurement of glucose cycling (see Ref. (7) for details) at 5 and 11 mM glucose concentrations. Cas9 expressing single cell clone from panel (B) was used as βTC3 wild-type (WT) control. Data represent means ± SEM, ∗∗∗p < 0.01, ∗∗p < 0.05 (n = 3). E, measurement of glucose-6-phosphatase (G6Pase) activity in vitro to further validate the knockout of G6pc2 in βTC3 cells. G6Pase activity from INS-1 cells, which do not express functional G6PC2, is included as a background control. Values represent the release of inorganic phosphates per min per mg of total protein. Data represent means ± SEM, ∗∗∗p < 0.01 (n = 3). KO, knockout.