rAAV-PV-GCaMp6m-Flag-WPRE-hGH polyA

Promoter Details

PV, GABAergic (inhibitory) neuron promoter, 2979/821bp.

Functional Gene Details

GCaMP: GCaMP consists of green fluorescent protein (GFP), calmodulin (CaM) and a peptide sequence M13 of myosin light chain kinase. When Ca2+ binds to CaM, CaM undergoes a conformational change, and its light chain region can bind to M13, causing the protonation of the chromophore of GFP to increase under illumination of a specific wavelength, and the absorbance increases, causing GFP to emit strong fluorescence. GCaMP6 is currently a conventional GECI, and its calcium indicating function reaches or even exceeds that of commonly used chemical calcium indicators. It has three commonly used subtypes, 6s, 6f and 6m. Among them, the binding force of calcium ions is 6s>6m>6f, so 6s is the most sensitive and suitable for the indication of low-frequency signals. 6f dissociates the fastest and is suitable for the indication of high-frequency signals. 6m has moderate binding activity and the widest range of application. GCaMP6 is an ultra-sensitive fluorescent protein that can specifically and efficiently reflect changes in intracellular free Ca2+ concentration. In vivo, GCaMP6 is 10 times stronger and kinetically 2 times faster than GCaMP3 for the calcium signal induced by a single action potential. Therefore, GCaMP6 fluorescent protein can be used as a hypersensitive Ca2+ probe to study the impact of external factors on Ca2+ in living cells under physiological and pathological conditions. The excitation wavelength is 495nm. GCaMP6s slow (100-150 ms) has the strongest light intensity and quenches later (most sensitive at low frequencies) GCaMP6m medium (75-100 ms) has medium light intensity and quenching (medium) GCaMP6f fast (50-75 ms) has weaker light intensity and quenches faster (high frequency)

Fluorescent Tag

Flag

Fluorescent Protein Details

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

Application

Measuring neuronal activity using genetically encoded calcium indicators (GECIs) has become a widely used method in neuroscience. Calcium imaging with GECIs is often used to measure neural activity in the complete nervous system, such as detecting the activity of a large number of neuronal cell bodies, or tracking the dynamics of neuronal subcells (such as axons, dendrites, and single synapses). Calcium imaging system is a type of optogenetic technology that uses calcium ion indicators that can sense Ca2+ concentration to detect Ca2+ concentration changes in cells or tissues and convert calcium concentration changes into fluorescent signals, thereby converting cell electrical activity into recordable light signals.

Tag Details

Flag: The Flag-tagged 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.