Figure 1. Design and validation of the targeted deletion of
ovalbumin distal promoter elements in DF1 cells.
- The schematic representation of CRISPR/Cas9 mediated deletion strategy
of the ovalbumin promoter in DF1 cells. The top diagram shows the
wild-type (WT) chicken ovalbumin locus. The two guide RNA (gRNA 1 and
2) binding sites are shown by red bars below the regulatory sequences
of the ovalbumin promoter. The gRNAs 1 and 2 target two positions
downstream of NRE (downstream of CAR; COUP-adjacent repressor site in
the negative regulatory element) and upstream of SDRE, respectively.
The bottom diagram shows the locus after CRISPR-mediated deletion of
the distal ovalbumin promoter in DF1 cells. The PCR primers used for
the assessment of deletion (P1 to P3), and the ovalbumin gene
expression (P4 and P5, used in Figure 2) are shown as small arrows.
- Two-step genomic PCR to confirm the deletion of distal promoter of the
ovalbumin gene. In the first PCR (using P1 and P3 primers), an
amplicon of 1310 bp was acquired from the wild-type (WT) allele. In a
hemi-nested PCR (using P1 and P2 primers), amplicons of 1256 bp and
~316 bp were acquired from the wild-type and
promoter-deleted (DF1 ∆) alleles, respectively.
- Alignment of the representative sequences of the wild-type (WT DF1)
and promoter-deleted (DF1 ∆) sequences acquired by
Sanger sequencing. The gRNA-binding sites regions are shown in blue,
and the PAM regions are shown in green letters. WT, wild type; DF1∆, distal ovalbumin promoter knockout DF1 cells (DF1+/OVA Pro ∆); NHEJ, non-homologous end-joining; ERE,
estrogen-responsive enhancer element; TSSL, tissue-specific
silencer-like element; SDRE, steroid-dependent regulatory element;
NRE, negative regulatory element; CAR, COUP-adjacent repressor site;
COUP, Chicken ovalbumin upstream promoter; TATA, TATA box; INR,
Initiator element; P, primer. M, DNA size marker; NTC, no template
control.