PFD-rAAV-EF1a-DO-hM4D(Gi)-mCherry-WPREs

Promoter Details

Ef1α, a strong promoter, human targeted elongation factor 1α (EF1A) gene, can be stably expressed in most types of cells, especially cells where the CMV promoter is silenced, such as stem cells, 1178bp.

Functional Gene Details

hM4D(Gi): It is an artificial receptor modified from the human muscarinic acetylcholine receptor M4 that only responds to CNO and is no longer activated by acetylcholine. In some neurons, CNO binds to hM4D(Gi) and activates the Gi protein-coupled inward potassium channel GIRK, hyperpolarizing the cell membrane and inhibiting the firing of action potentials in neuronal cells. In cells that do not express GIRK or Gi, hM4D(Gi) functions differently.

Fluorescent Tag

mCherry

Fluorescent Protein Details

mCherry: red fluorescent protein, excitation wavelength 587nm, emission wavelength 610nm.

Application

Chemical genetics technology/DREADDS (designer receptors exclusively activated by designer drugs) technology can be used to regulate the activities of neurons or other types of cells. For example, by injecting the virus into a specific brain area of ​​a mouse, and after hM4D(Gi) is expressed in the mouse, CNO (clozapine-N-oxide, N-oxide clozapine) is given via intraperitoneal injection or diet, which can activate neurons over a long period of time (on the order of hours). DREADDs technology can be used to explore neurons and neural circuits related to memory formation, food intake regulation and arousal.

Regulatory Element Details

DO: The inserted gene is in the same direction as the promoter. In the presence of Cre recombinase, recombination will occur, causing the gene direction to be reversed, so the gene is not expressed, so it is called Cre-Off.

Notes

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 time it is frozen and thawed, the titer will be reduced by 10%, and 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.