Zebrafish is an attractive vertebrate model for analyzing the structure and function of neural circuits because of its small size, transparency in early development, genetic modification, and suitable for electrophysiological and optical measurement of neuronal activity. Zebrafish embryos with optogenetic elements can be obtained by transgenic means. However, zebrafish does not provide an effective method for rapid neuronal gene transfer in vivo at the post embryonic stage.
Creative BioMart provides two optimized programs to directly introduce herpes simplex virus type I (HSV-1) or electroporation into the adult zebrafish brain to express multiple channelrhodopsin-2 variants and genetically encoded calcium indicators in telencephalon neurons for measuring neuronal activity and synaptic connections.
Viral vectors have practical disadvantages, including toxicity, complex procedures for virus production and modification (rabies, baculovirus) and difficulty in producing high titers (rabies). One possibility to circumvent these problems is modified herpes simplex virus 1 (HSV-1).
HSV-1 can produce dense expression of transgene, which is important for targeting a large number of neurons. The ability of HSV-1 to retrogradely infect neurons through axons can be used to target defined projection neurons. It is conceivable that additional cell type selectivity can be generated by selecting promoters, which can be exchanged using established procedures.
Using a stereotactic procedure, HSV-1 virus was injected into the dorsal telencephalon, DP or olfactory bulb through small craniotomy (method), and HSV-1 virus was injected into fish.
Most of the fluorescence produced by HSV-1 virus can be observed through the intact skull. Fluorescence was first observed 2 days after injection and usually lasted for more than 4 weeks. We can use CMV promoters for short-term or long-term expression according to customer's requirement.
Electroporation is a method of transiently permeating the plasma membrane and transferring nucleic acids into cells using a short electric pulse.
One of the advantages of electroporation is that plasmids can be introduced directly into neurons without packaging genetic material into viruses or other vectors. Therefore, the time to produce reagents for gene transfer and the risk of immune response or other potential complications are reduced. In addition, the efficiency of electroporation should not vary greatly between cell types, brain regions and even species, because electroporation depends on physical mechanisms rather than molecular mechanisms. Therefore, electroporation is a particularly rapid and universal gene transfer method.
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