Optogenetics

rAAV-CAG-DIO-OChIEF-mCherry-WPREs

The broad-spectrum promoter CAG regulates the expression of Cre-dependent light-activated elements. OChIEF-mCherry is expressed when Cre is present, but OChIEF-mCherry is not expressed when Cre is absent. Optogenetic activation product instructions are for scientific research use only. Please read the instructions carefully before use. rAAV: Recombinant adeno-associated virus, recombinant adeno-associated virus. Using recombinant adeno-associated virus as a vector, it has high safety (rAAV has not yet been found to be pathogenic to humans), low immunogenicity (rarely induces an immune response after infection), has a long expression time, and can infect a variety of cells and tissues. Virus use: rAAV viral vectors are used to express light-sensitive ion channel proteins, such as ChR2, ChrimsonR, etc., in neurons to specifically excite neurons and explore the effects of light stimulation on neurons, neural circuits and animal behavior. Promoter: CAG, strong promoter, artificially constructed combined promoter, composed of cytomegalovirus early enhancer and chicken β-actin promoter, 1731bp. Functional gene: OChIEF: ChIEF is a fusion of two proteins, ChR1 and ChR2, and Ile at position 170 is mutated to Val. The characteristic of this protein is that under long-term illumination, the ratio of plateau current to peak current is large, which means that the channel is less inactivated under illumination and is suitable for long-term stimulation. The mutation of Ile accelerates the closing speed of the channel, making ChIEF suitable for high-frequency stimulation. Karl's laboratory has compared the high-frequency stimulation of ChIEF and hChR2(E123A) in pv (small serum protein) neurons, and the effects are equivalent. OChIEF is a codon-optimized version. The excitation light wavelength is 473nm, suitable for high-frequency light activation. Fluorescent protein: mCherry: red fluorescent protein, excitation wavelength 587nm, emission wavelength 610nm. Regulatory element: DIO: Double-floxed inverse Orientation, which is an element that cooperates with Cre recombinase to control the expression of the target gene. If there is no Cre recombinase, the target gene will not be expressed, but in the presence of Cre, the target gene can be expressed normally. Note: 1. Short-term storage at 4°C; long-term storage at -80°C; 2. It can be stored at -80°C for more than 6 months. Each freeze and thaw will reduce the titer by 10%. Repeated freezing and thawing should be avoided; if the virus is stored for more than 6 months, it is recommended to re-measure the virus titer before use; 3. According to the experimental needs, the virus can be appropriately diluted and divided into the volume required for each experiment.