rAAV-CaMKIIa-DIO-hM3D(Gq)-3XFlag-WPREs

Promoter Details

CaMKIIa, pyramidal neuron/excitatory neuron promoter, 1293bp.

Functional Gene Details

hM3D(Gq): It is an artificial receptor modified from the human muscarinic acetylcholine receptor M3 that only responds to CNO and is no longer activated by acetylcholine. In some neurons, after CNO binds hM3D(Gq), it activates the Gq protein-coupled phosphatase PLCβ, causing PIP2 (phosphatidylinositol(4,5)bisphosphate, phosphatidylinositol diphosphate) to be degraded, thereby opening the KCNQ outward potassium ion channel closed by PIP2, so that the cell membrane is depolarized, forming an action potential, that is, activating neurons. In neurons that do not express KCNQ or Gq, the function of hM3D(Gq) may be different.

Fluorescent Tag

3XFlag

Fluorescent Protein Details

EGFP: Green fluorescent protein, excitation wavelength 488nm, emission wavelength 507nm.

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 hM3D(Gq) 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

DIO: Double-floxed inverse Orientation, 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. 3XFlag: The Flag tag protein is a hydrophilic polypeptide encoding 8 amino acids (DYKDDDDK). At the same time, the Kozak sequence constructed in the vector makes the fusion protein with FLAG more efficient in eukaryotic expression systems. The target protein fused to FLAG can be directly purified by non-denaturing affinity chromatography through FLAG to purify the active fusion protein with high purification efficiency. Fusion proteins containing FLAG can also be detected and identified through methods such as Westem Blot and ELSA. FLAG fused to the N-terminus can be cleaved by enterokinase (DDDK) to obtain the specific target protein.

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.