Fig. 4

Activation of transgene expression in DF1 ^{+/OVA Pro ∆−Tg (promoterless dsRed)} cells. A The schematic representation of CRISPR HDR mediated knockin strategy in DF1^{+/OVA Pro ∆} cells. The top diagram shows the donor vector that was designed to have a promoterless DsRed2 and a CMV-Puro-EGFP cassette flanked by left and right homology arms. The OVA E2 indicates the gRNA-binding site on exon 2 of the OVA (+ 174 to + 1784) gene. The bottom diagram shows the allele after CRISPR-HDR insertion of the reporter cassette (DF1 ^{+/OVA Pro ∆−Tg (promoterless dsRed)}). PCR primers (P12 and P13) were used for the assessment of the CRISPR-HDR insertion of the promoterless DsRed2 in DF1 ^{+/OVA Pro ∆−Tg (promoterless dsRed)} cells. B Genomic PCR analysis of the targeted gene knock-in DF1 ^{+/OVA Pro ∆−Tg (promoterless dsRed)} cells. For the assessment of the CRISPR-HDR insertion of the promoterless DsRed2 in DF1 ^∆−Tg cells, primers (P12 and P13) were used to amplify a 2569 bp amplicon. The insertion-specific PCR products of DF1 ^∆−Tg cells were sequenced by Sanger sequencing and aligned to the donor plasmid (used as a DNA repair template during transfection). C Fluorescence microscopy of DF1 ^∆−Tg cells indicating DsRed2 expression under the control of the endogenous truncated OVA promoter, Magnification: 20X. DF1^∆, DF1 cells knock-out for distal OVA promoter (DF1 ^{+/OVA Pro ∆}); DF1 ^∆−Tg cells, promoterless DsRed2 knockin DF1 cells (DF1 ^{+/OVA Pro ∆−Tg (promoterless dsRed)}); HDR, homology-directed repair; M, DNA size marker; WT, wild-type; NTC, no template control