PFD-rAAV-hSyn-axon-jGCaMP7b-WPRE-hGH polyA

Promoter Details

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

Functional Gene Details

jGCaMP7: By optimizing the Ca2+ affinity of a calcium ion probe while further improving the probe's sensitivity, signal-to-noise ratio, half-life, and dynamic range, Karel Svoboda, Loren Looger, and Vivek from the Howard Hughes Medical Institute Jayaraman and other research groups jointly developed the jGCaMP7 series, including: jGCaMP7s (sensitive), jGCaMP7f (fast kinetics), jGCaMP7b (brighter baseline fluorescence) and jGCaMP7c (high contrast), which are specially used to detect neuronal activity in specific environments. Tracking the dynamics of neuronal subcells is of great significance to the study of neural circuits. However, subcellular structures such as dendrites and axons are small, and jGCaMP6 has low sensitivity and low imaging fidelity. However, jGCaMP7s, jGCaMP7f, and jGCaMP7b have greatly improved the sensitivity under different calcium ion concentrations. jGCaMP7f also has a faster response rate, which is very important for the dynamic detection of neuronal subcells. jGCaMP7 was tested both in vivo and in vitro and performed significantly better than GCaMP6. The sensitivity of jGCaMP7s is more than 5 times that of GCaMP6s, and the speed is faster than GCaMP6s; the sensitivity of jGCaMP7b is 3 times that of GCaMP6s, and the fluorescence brightness is enhanced by 50%; the sensitivity of jGCaMP7c is 2.7 times that of GCaMP6s, and the contrast is enhanced; the reaction rate of jGCaMP7f is 5 times that of GCaMP6f and 3 times that of GCaMP6s. jGCaMP7s is more sensitive jGCaMP7f fast dynamics jGCaMP7b high background jGCaMP7c high contrast, low background The N-terminus of jGCaMP7 protein is coupled to a targeting element GAP43 (growth associated protein-43) that drives axon positioning, thereby achieving axon- jGCaMP7 is expressed in dendrites and distal axons, so it can detect changes in calcium ion concentration in axons.

axon-GCaMP: The Axon-GCaMP6 system is composed of the N-terminus of the conventional GCaMP protein coupled to a targeting element GAP43 (growth associated protein-43) that drives axon positioning, thereby realizing the expression of axon-GCaMP in dendrites and distal axons, so it can detect changes in calcium ion concentration in axons. 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.

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.