rAAV-CaMKIIa-HA-KORD-IRES-mCitrine-WPRE-hGH polyA

Promoter Details

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

Functional Gene Details

KORD: kappa-opioid receptor based DREAAD, which is an artificial receptor modified from the human kappa opioid receptor and is sensitive to SALB (serum albumin) and is not activated by ligands such as opioids. SALB has good blood-brain barrier permeability and has no obvious effect on animals at physiological doses. After SALB binds to KORD, it activates Gi-coupled effector proteins, increases the inward potassium ion current, causes cell membrane hyperpolarization, and inhibits action potential generation. In different cells, the functions of KORD will vary.

Fluorescent Tag

mCitrine/HA

yellow fluorescent yellow

Fluorescent Protein Details

mCitrine: yellow fluorescent protein, excitation wavelength 516nm, emission wavelength 529nm. Tag protein: HA: The HA tag protein sequence is YPYDVPDYA, which is derived from the hemagglutinin surface epitope of influenza virus. It has 9 amino acids and has little impact on the spatial structure of the foreign target protein. It is easy to construct a tag protein fused to the N-terminus or C-terminus. Easy to detect with Anti-HA antibody detection and ELISA. IRES: internal ribosome entry site, the Chinese name is the internal ribosome entry sequence, which can be inserted downstream of the CDS region on the mRNA to mediate another CDS region that follows it, thus enabling the expression of two proteins on one mRNA.

Virus Type

Adeno-associated virus vector Adeno-associated Virus

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.

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.

Expression and Regulation Mode

specific expression