Fig. 3

Gene expression ratio for Ovalbumin transcript in DF1^{+/OVA Pro ∆} cells. A Agarose gel (2%) electrophoresis for analysis of the RT-PCR products amplified by primers P8 and P9 (for OVA, Fig. 1), and P10 and P11 (for GAPDH). The expected amplicon size for OVA and GAPDH are 179 bp and 187 bp, respectively. WT, wild-type; DF1 ^∆, distal OVA promoter knockout DF1 cells (DF1 ^{+/OVA Pro ∆}); M, DNA size marker; NTC, no template control; RT, reverse transcriptase. The full-length gel electrophoresis images are shown in Fig. S3. B Upregulation of the OVA mRNA in DF1 ^{+/OVA Pro ∆}cells was assessed by RT-qPCR. Upon deletion of the distal OVA promoter, an increased level of expression of the OVA gene was determined (DF1^∆). The transcript levels of OVA for these samples (Three isogenic DF1 ^{+/OVA Pro ∆} clones) were ~ 10⁴-fold higher than the OVA transcript levels in the wild-type DF1 (WT DF1). The transcript levels for OVA in the hormonally-activated tissue of the magnum (from a 35-week-old laying hen) show the highest level of expression. The gene expression ratio for the OVA over GAPDH was calculated by the Pfaffl method of relative quantification [38]. The Mann–Whitney assay was used to analyze significant statistical differences between the WT-DF1 group and DF1^∆ and magnum groups. * and ** show statistical differences with p values < 0.05 and < 0.01, respectively