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. 2018 Nov 29;13(11):e0201683. doi: 10.1371/journal.pone.0201683

Fig 2. Transient puromycin selection increases reporter KI efficiency.

Fig 2

(A) Schematic of gene reporter design created by CRISPR editing. (B) Phase and fluorescence microscopy images of TBP-P2A-eGFP KI into H9 hESCs after puromycin selection. Scale bar = 100 μm. (C) Representative images of flow cytometry assessment of TBP-P2A-eGFP KI into H9 hESCs with and without transient puromycin selection. Untransfected cells were used to set the gates for reporter negative cells. Two peaks of eGFP+ cells can be observed in the puromycin treated group suggesting homozygous and heterozygous KI. (D) Flow cytometry analysis of TBP-P2A-eGFP, MYC-P2A-eGFP, and SOX2-P2A-eGFP KI into H9 hESCs with and without transient puromycin selection. For TBP n = 2 for both groups, for MYC n = 2 for replicates without puromycin and n = 5 for replicates with puromycin treatment, for SOX2 n = 3 for replicates without puromycin and n = 4 for replicates with puromycin treatment. n = biological replicates. p values: TBP = 0.0047, MYC = 0.0041, SOX2 = 0.0057. ** = p < .01. Unpaired two tailed t-test was used.