Adeno associated virus (AAV) belongs to the parvovirus family because its single stranded DNA genome does not contain envelope. After receptor-mediated endocytosis in the somatic compartment and transfer to the nucleus, the transgene encoded protein is synthesized, transported anterogradely to the axon end and integrated into the membrane to allow the optogenetic regulation of axon projection. For AAV of various serotypes, retrograde transport of AAV particles from nerve endings to cell bodies is also possible.
Figure 1: Workflow of AAV production for optogenetic studies. (Thompson K.R., 2018) AAV has become the preferred vector for optogenetic research in rodents because it has higher neurotropism and relatively low inflammatory response compared with other vectors. The main limitation of AAV is its relatively small packaging capacity, which is not the main obstacle, because the size of microbial opsin is easily suitable for recombinant viral genome.
Creative BioMart provides a simple and reliable method to produce high-quality AAV of any serotype in a short time using ready-made materials.
The opsin of interest was cloned into the AAV backbone between reverse terminal repeats (ITRS) to create opsin particles. Recombinant AAV was produced by triple transfection of opsin particles with AAV structural plasmid and adenovirus helper plasmid into 293 cells. 48 hours later, the cell homogenate was loaded onto the iodixanol gradient and centrifuged to separate AAV from cell fragments. AAV was then titrated and quality controlled before stereotactic injection into the rodent brain.
| Step | Service Description |
| Cloning, cell culture and transfection | The new opsin gene can be directly cloned into pAAV: MCS expression plasmid by conventional molecular biology technology 293 T cells were maintained with growth medium in a humidified incubator with 5% CO2. |
| AAV isolation | 4 days after transfection, the recombinant AVV virus was isolated by standard method Purified by iodixanol gradient AAV Storage of recombinant AVV |
| AAV titration and quality control | There are various protocols to measure the characteristics of the produced recombinant AAV vector. The titration method reports the dose of the carrier in many different ways. Quantitative PCR titer In vitro infection titer SDS-PAGE for purity analysis |
| In vivo delivery | After production and quality control, the opsin carrier expressing AAV is ready to be introduced into vivo for optogenetic research. AAV can be injected directly into the brain. |
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