Locust is a widely used animal model to study sensory processing and its relationship with behavior. Locust is a popular model to study behavior dependent on visual movement, especially visual induced escape and collision avoidance behavior. It is desirable to create transgenic locust strains or develop effective transfection methods in locusts to provide neuronal activity indicators or regulators to improve the ability of the model system. Photogenetics is an effective stimulation method, and optical gated ion channels (such as rhodopsin, halorhodopsin and their variants) can be used to control neuronal activity.
Semliki Forest virus has been proved to be suitable for mediating the expression of foreign genes in locust optic lobe neurons. Using Semliki Forest virus, the channel rhodopsin can be effectively transported to the neurons of locust optic lobe. As an unparalleled global provider of life science services and products, Creative BioMart provides one-stop services covering all steps of vector construction. We subcloned channelrhodopsin variant and yellow fluorescent protein Venus into Semliki Forest virus vector. The virus vector can be injected into the optic leaves of locusts (American schizococci).
We inserted a variant of channelrhodopsin into the SFV A7 based vector. The means of vector construction depends on the expression plasmid encoding SFV non structural protein and foreign gene (transgenic) , while the second plasmid is used to synthesize the required viral structural protein for packaging SFV particles. After in vitro transcription of vector and auxiliary RNA, both RNAs were electroporated into secondary tissue cultures for SFV protein synthesis and virus assembly.
Since the virus packaging signal exists in the vector rather than in the auxiliary RNA, only the vector RNA is packaged into the recombinant virus particles. After subsequent infection of target cells, the replicon will replicate its viral RNA genome without producing new infectious particles. As an additional safety feature of SFV vector, virus particles are designed to be conditionally infected because their activation requires the cleavage of non physiological high concentration chymotrypsin, i.e. spike precursor protein.
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