PFD-rAAV-hSyn-DIO-NLS-GCaMP6s-WPRE-hGH polyA

Promoter Details

hSyn, human synapsin promoter, mature neuron-specific promoter, 448bp.

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)

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.

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. Localization sequence: NLS: Nuclear Localization Signal, nuclear localization signal peptide sequence. The most important signal peptide sequences include the pelB leader sequence and SV40 NLS, among which pelB is a signal peptide that targets expressed proteins to the periplasmic space, while SV40 NLS is the most commonly used eukaryotic cell nuclear localization signal. For example, the N-terminal or C-terminal of the Cas9 gene in the commonly used CRISPR series vectors will be fused to this sequence.

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.