rAAV-EF1a-fDIO-hM4D(Gi)-EGFP-3XFlag-WPRE-hGH polyA

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

EGFP/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 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

fDIO: It is an element that cooperates with Flp to control the expression of target genes. If there is no Flp recombinase, the target gene will not be expressed. Only in the presence of Flp can the target gene be expressed normally. 3XFlag: The Flag tag protein encodes a hydrophilic polypeptide of 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.