3.1.Deletion of the ovalbumin distal promoter elements in DF1 cells via CRISPR/Cas9 system

Previous studies have shown that the cloning of proximal segment of the ovalbumin promoter lacking the major regulatory sequences of SDRE and NRE is able to increase the chloramphenicol acetyltransferase (CAT) activity on a plasmid construct in LMH cells and primary oviduct cells (Haecker et al., 1995; Monroe & Sanders, 2000). This activity was not dependent on the presence of estrogen. However, the observed increased activity was up to 17-fold. Thus, we decided to delete SDRE and NRE elements from the native promoter in the genome of a non-oviduct cell. To this end, we used CRISPR excision strategy to delete regulatory elements from the ovalbumin promoter in DF1 fibroblast cells (Figure 1A). Cells with the deleted promoter segments were subjected to genomic PCR and Sanger sequencing to confirm the deletion (Figures 1B and 1C). Then, 288 cells were individually grown to acquire clones with homogenous population of cells.

3.2.Deletion of the ovalbumin distal promoter elements induces the expression of ovalbumin mRNA

We asked whether the deletion of SDRE and NRE elements from the native promoter in the genome of a non-oviduct cell would be able to increase the Ovalbumin transcription. Thus, three individually grown DF1 clones (from 288 clones) with the deletion of SDRE and NRE elements were analyzed for the expression of Ovalbumin by RT-qPCR. In DF1 cells lacking the SDRE, linker and NRE segments in their ovalbumin promoter and grown in vitro in the absence of estrogen, the transcript levels of Ovalbumin increased to more than 104-fold compared to that in the wild-type DF1 cell. The transcript levels of Ovalbumin in the hormonally-activated tissue of magnum from 35-week old laying hen was 107-fold compared to that in the wild-type DF1 cells (Figure 2).

3.3.The inserted fluorescent reporter is activated under the control of the ovalbumin promoter with the deletion of distal elements

Next, we tried to assess the functionality of the distally-deleted ovalbumin promoter in DF1 cells to activate a foreign transgene. For this purpose, we designed a construct including a promoterless reporter and used CRISPR HDR to insert it in the exon 2 of the Ovalbumin gene (125 bp after ATG) (Figure 3A). The insertion of this reporter was confirmed by genomic PCR, Sanger sequencing, and fluorescence microscopy for GFP (Figures 3B, 3C, and the left panel in 3D). The promoterless DsRed2 reporter under the function of distally-deleted ovalbumin promoter became activated and its red fluorescence was imaged by fluorescence microscopy (Figure 3D). This experiment confirmed that a non-oviduct chicken cell with the deletion of distal elements of ovalbumin promoter can express an inserted transgene in an estrogen-independent manner.