rAAV-CAG-FLEX-hChR2(H134R)-HA-HA-T2A-hM4D(Gi)-WPRE-SV40 polyA

Promoter Details

CAG, strong promoter, an artificially constructed combined promoter, composed of cytomegalovirus early enhancer and chicken β-actin promoter, 1731bp.

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. hChR2(H134R): It is the most common form of ChR2. h refers to the coding sequence of the gene that has been optimized for the human codon table. H134R refers to the mutation of histidine at position 134 to arginine. This mutation inhibits the binding of H+ by ChR2 and increases the passing ability of Na+, thereby increasing the overall conductivity of the molecule and thereby increasing the inward current excited by light. Generally, blue light around 470nm is used for excitation.

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

HA

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

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

FLEX: DIO and FLEX are very close, except for the interval 1 and interval 2 sequences used. DIO is facing inward, and Flex is facing outward. 2A: The 2A sequence is a short peptide sequence. When the ribosome translates it, it will automatically jump away and continue to translate the downstream reading frame, thereby translating the same reading frame into two independent proteins. Used in non-fusion expressions. Commonly used 2A sequences include T2A and P2A. 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.

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.