| Trichoplusia ni (T. ni) larvae are low cost alternatives to insect or mammalian cell culture or bacteria fermentation. They are easy to grow, they are 10--100 times less expensive than insect cell culture, they have their own immune systems that reduce the effects of contamination, they can perform many of the posttranslational modifications necessary to produce active enzymes, and scale-up of the system is linear. One disadvantage of the T. ni larval system is that the endogenous proteases produced by the larvae for general cellular function, molting, and digestion of food can also degrade recombinant proteins.; We are exploring the use of RNA interference (RNAi) to reduce this endogenous protease activity. RNAi refers to any RNA molecule that can inhibit production of protein from a homologous target sequence. This includes sense strand RNA, antisense-strand RNA, and double-stranded RNA (dsRNA). We have produced RNAi to UV-optimized green fluorescent protein (GFPuv) in vitro and injected it into T. ni larvae infected with a baculovirus (AcMNPV: Autographa californica multiple nucleocapsid nuclear polyhedrosis virus) encoding GFPuv under the control of the polyhedrin promoter. RNAi inhibited the GFPuv production, and this inhibition was dependent on the time the RNAi was injected into the larvae relative to the baculovirus infection, and on the concentration of the injected RNAi. These results indicate that RNAi can be used in T. ni larvae. To further explore the potential of RNAi for the inhibition endogenous of proteins in T. ni larvae, RNAi was made in vitro to larval genes, such as heat shock proteins, metamorphosis-related proteins, and/or larval proteases, and the effects on recombinant protein production was quantified. RNAi to endogenous larval genes caused morphological and behavioral changes in the larvae. Additionally, coinjection of RNAi to various larval genes and a baculovirus expressing GFPuv changed the expression levels of the GFPuv protein. This indicates that RNAi can be used as a metabolic controller in T. ni larvae to manipulate host functions and increase recombinant protein production. |
