5 PRODUCTION OF TRANSGENIC LIVESTOCK USING A LENTIVIRUS EXPRESSING MULTIPLE SHORT INTERFERING RNAs TARGETING FOOT AND MOUTH DISEASE VIRUS

Abstract

One goal of transgenic livestock production is developing animals with enhanced production characteristics. Transgenic animals with resistance to viral disease could greatly reduce economic losses. The use of short interfering RNA (siRNA) or short hairpin RNA (shRNA) targeting viral genomes have shown great promise in vitro for both human and animal applications. However, because of the rapid mutation rate, viruses are able to escape single siRNA inhibition. One method to reduce the chances of a functional escape virus is to target its genome with multiple shRNAs simultaneously. The goal of this research project was to produce a recombinant lentiviral vector that expresses three unique shRNAs targeting different regions of the foot and mouth disease virus (FMDV) and use it to produce transgenic livestock. In these initial experiments we used the goat as our model system. Previously, we confirmed that three distinct siRNAs individually could reduce the ability of the FMDV virion to replicate in vitro. Based upon these results we produced a recombinant lentiviral vector that utilised three bovine Pol III promoters (7sk, H1, U6-2), each transcribing a different effective shRNA targeting FMDV. In addition, the vector also contained the fluorescent marker zsGreen and an antibiotic resistance gene. The lentiviral vector was co-transfected with pCMV and pMDG into 293T cells to produce replication incompetent retroviral particles. The supernatant was collected and ultra-centrifuged (50 000 × g for 1.5 h) to concentrate the viral particles resulting in a high-titer viral preparation (>10⁹ mL^–1 infective viral particles). To produce the transgenic caprine offspring, three embryo donors were superovulated and naturally bred. Nineteen zygotes were surgically collected from the oviduct 24 h after mating. Recombinant lentivirus was microinjected into the zygote perivitelline space. Immediately following the injections, four goat embryos were surgically transferred into the oviduct of each synchronized recipient. Pregnancy was determined by ultrasound at Day 30 in 2 of 5 recipients that received embryos. One pregnancy was carried to term resulting in triplets; 1 live birth, and 2 stillborn. The placenta and tissue sample of the live goat both contained a subpopulation of zsGreen positive cells when analysed with fluorescent microscopy. A fibroblast cell line was derived from the tissue sample and placed under antibiotic selection. Results indicate that only the fluorescent cells also expressed a resistance to antibiotic selection. RNA was collected from the fibroblast cells and mature shRNA production was confirmed using the QuantiMir kit (System Biosystems). Expression of all 3 mature shRNAs was verified in these cells. This data further supports that the entire transgene was integrated into the genome. This is the first report of transgenic livestock produced that expresses multiple shRNAs targeting a viral genome.