Abstract
Farm animal salivary glands hold great potential as efficient bioreactors for production of human therapeutic proteins. Nerve growth factor (NGF) is naturally expressed in animal salivary glands and has been approved for human clinical treatment. This study aims to employ transgenic (TG) pig salivary gland as bioreactors for efficient synthesis of human NGF (hNGF). hNGF-TG pigs were generated by cloning in combination with piggyBac transposon-mediated gene transfer. These hNGF-TG pigs specifically expressed hNGF protein in their salivary glands and secreted it at high levels into saliva. Surgical and nonsurgical approaches were developed to efficiently collect saliva from hNGF-TG pigs. hNGF protein was successfully purified from collected saliva and was verified to be biologically active. In an additional step, the double-transgenic pigs, where the endogenous porcine NGF (pNGF) gene was replaced by another copy of hNGF transgene, were created by cloning combined with CRISPR/Cas9-mediated homologous recombination. These double-transgenic pigs expressed hNGF but not pNGF, thus avoiding possible "contamination" of hNGF with pNGF protein during purification. In conclusion, TG pig salivary glands can be used as robust bioreactors for a large-scale synthesis of functional hNGF or other valuable proteins. This new animal pharming method will benefit both human health and biomedicine.