No abstract

The salivary glands of animals have great potential to act as powerful bioreactors to produce human therapeutic proteins. Human nerve growth factor (hNGF) is an important pharmaceutical protein that is clinically effective in the treatment of many human neuronal and non-neuronal diseases. In this study, we generated 18 transgenic (TG) founder mice each carrying a salivary gland specific promoter-driven hNGF transgene. A TG mouse line secreting high levels of hNGF protein in its saliva (1.36 μg/mL) was selected. hNGF protein was successfully purified from the saliva of these TG mice and its identity was verified. The purified hNGF was highly functional as it displayed the ability to induce neuronal differentiation of PC12 cells. Furthermore, it strongly promoted proliferation of TF1 cells, above the levels observed with mouse NGF. Additionally, saliva collected from TG mice and containing unpurified hNGF was able to significantly enhance the growth of TF1 cells. This study not only provides a new and efficient approach for the synthesis of therapeutic hNGF but also supports the concept that salivary gland from TG animals is an efficient system for production of valuable foreign proteins.

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.

About 70% of all antibiotics produced in the world are used in the farm animal industry. The massive usage of antibiotics during farm animal production has caused rapid development of antibiotic resistance in bacteria, which poses a serious risk to human and livestock health when treating bacterial infections. Protegrin-1 (PG-1) is a potent antimicrobial peptide (AMP). It was initially identified in pig leukocytes with a broad-spectrum antibacterial and antiviral activity, and a low rate of inducing bacterial resistance. To develop a genetic approach for reducing the use of antibiotics in farm animal production, we produced transgenic mice carrying a bovine tracheal AMP gene promoter-controlled PG-1 transgene. The PG-1 transgene was specifically expressed in the respiratory tract of transgenic mice upon induction by bacterial infection. These PG-1 transgenic mice exhibited enhanced resistance to nasal bacterial infection as the transgenic mice showed a higher survival rate (79.17% VS. 34.78%), lower bacterial load and milder histological severity than their wild-type control littermates. The improved resistance to bacterial infection in the PG-1 transgenic mice could be resulted from the direct bacteria-killing activities of PG-1, and the immunomodulatory effects of PG-1 via stimulating interleukin 1 beta secretion. The present study provides a promising genetic strategy to prevent airway bacterial infections in farm animals by bacteria-inducible tissue-specific expression of PG-1 transgene. This approach may also be helpful for decreasing the possibility of inducing bacterial resistance during farm animal production.