Sequential verification of exogenous protein production in OVA gene-targeted chicken bioreactors

The chicken has potential as an efficient bioreactor system because of its outstanding protein production capacity and low cost. The CRISPR/Cas9-mediated gene-editing system enables production of highly marketable exogenous proteins in transgenic chicken bioreactors. However, because it takes approximately 18 mo to evaluate the recombinant protein productivity of the bioreactor due to the generation interval from G0 founders to G1 egg-laying hens, to verification of the exogenous protein at the early stage is difficult. Here we propose a system for sequential validation of exogenous protein production in chicken bioreactors as in hatching female chicks as well as in egg-laying hens. We generated chicken OVALBUMIN (OVA) EGFP knock-in (KI) chicken (OVA EGFP KI) by CRISPR/Cas9-mediated nonhomologous end joining at the chicken OVA gene locus. Subsequently, the estrogen analog, diethylstilbestrol (DES), was subcutaneously implanted in the abdominal region of 1-wk-old OVA EGFP KI female chicks to artificially increase OVALBUMIN expression. The oviducts of DES-treated OVA EGFP KI female chicks expressed OVA and EGFP at the 3-wk-old stage (10 d after DES treatment). We evaluated the expression of EGFP protein in the oviduct, along with the physical properties of eggs and egg white from OVA EGFP KI hens. The rapid identification and isolation of exogenous protein can be confirmed at a very early stage and high-yield production is possible by targeting the chicken oviduct.