Figure 3. Activation of transgene expression in DF1+/OVA Pro ∆-Tg (promoterless dsRed) cells.
  1. The schematic representation of CRISPR HDR mediated knockin strategy in DF1+/OVA Pro ∆ cells. The top diagram shows the donor vector that designed to have a promoterless DsRed2 and a CMV-Puro-EGFP cassette flanked by left and right homology arms. The gRNA3 (denoted by the red bar) indicates the gRNA-binding site on exon 2 of the ovalbumin (+174 to +1784) gene. The bottom diagram shows the allele after CRISPR-HDR insertion of the reporter cassette (DF1∆-Tg cells). PCR primers (P6 and P7) were used for the assessment of the CRISPR-HDR insertion of the promoterless DsRed2 in DF1 ∆-Tg cells.
  2. Genomic PCR analysis of the targeted gene knockin DF1∆-Tg cells. For the assessment of the CRISPR-HDR insertion of the promoterless DsRed2 in DF1 ∆-Tgcells, primers (P6 and P7) were used to amplify a 2569 bp amplicon in DF1 ∆-Tg cells.
  3. The insertion-specific PCR product of DF1 ∆-Tg cells was sequenced by Sanger sequencing and aligned to the donor plasmid (used as a DNA repair template during transfection).
  4. Fluorescence microscopy of DF1 ∆-Tg cells indicating DsRed2 expression under the control of the endogenous truncated ovalbumin promoter. DF1 , distal ovalbumin promoter knock-out DF1 cells (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.