Figure 3. Activation of transgene expression in DF1+/OVA Pro ∆-Tg (promoterless dsRed) cells.
- 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.
- 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.
- 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).
- 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.