Supplementary Figure 7: Characterization of CRISPR/Cas9-mediated GLUT1 knockout and negative control 4T1-luc cells.
From: Bioluminescent-based imaging and quantification of glucose uptake in vivo
(a) The scheme depicts the part of the Slc2a1 gene between exons 3 and 6, surrounding the targeting regions (named, A, B and C) of CRISPR/Cas9 plasmids that are a mixture of three gRNAs (Supplementary Table 1). Targeting regions were amplified using two sets of primers: primers oCF and oCR for region C, and primers oABF and oABR for region AB. Representative sequencing results of TA cloned PCR fragments from a parental cell line (4T1-luc), two negative non-targeting gRNAs control clones (NC no. 1, NC no. 2) and two knockout clones (GLUT1-/- no. 1, GLUT1-/- no. 2) are shown. In total, 10 colonies per region per clone were sequenced. There was no change observed in the sequence of NC clones compared to the parental control line. GLUT1 knockout clones harbored knockout alleles with frameshift mutation resulting from NHEJ. In addition, one knockout allele in the GLUT-/- no. 1 clone resulted in a deletion of 1062 bp between the region C and regions AB. (b) Summary table of sequencing results showing mutations in the targeted regions. (c) Western blot analysis of GLUT1 expression in 4T1-luc cells, 4T1-luc NC (no. 1, no. 2) and 4T1-luc- GLUT1-/- (no. 1, no. 2). For gel source images, see Supplementary Fig. 15. All experiments were performed independently at least twice.
