In ovo injection of antisense Cyp19a1 oligonucleotide for female-to-male sex reversal in chicken embryos

Chicken gene-editing is still challenging because only multicellular blastocysts are accessible in newly laid eggs. In this experimental study, we assessed the possibility of DNA uptake by day 2.5 embryos. We overlaid lipoplexes including a reporter plasmid encoding Venus fluorescent protein on top of embryos (n=11). Expression of the transgene was detected in 5 out of 7 developed embryos (71.4 %, day 5), indicating the ability of chicken embryos to absorb exogenous DNA. Then, we used antisense oligonucleotide (ASO) against Cyp19a1 to assess the possibility of female-to-male sex reversal. The ASOs were in ovo injected with or without IGF1 in the egg albumen of day 2.5 chicken embryos. In addition, as a positive control for sex reversal, we injected letrozole, an anti-aromatase, with/without IGF1. Using 0.5 mM ASO, a 44.4 % female-to-male sex-reversal was achieved. A combination of 0.5 mM ASO and IGF1 improved the sex reversal rate to 77.8 %. A synergistic effect of letrozole and IGF1 was observed for the female-to-male sex reversal. In conclusion, this study confirms DNA uptake by the chicken embryos and showcases the feasibility of in ovo gene knock-down using antisense oligonucleotides.