PFD-rAAV-CaMKIIa-ChrimsonR-P2A-GCaMP6f-WPREs

Additional Categories

Calcium Imaging

Promoter Details

CaMKIIa, pyramidal neuron/excitatory neuron promoter, 1293bp.

Functional Gene Details

ChrimsonR: A new type of light-controlled ion channel that can respond to red light around 600nm was discovered through high-throughput sequencing in the Boyden laboratory. A point mutation of K176R was made on the basis of Chrimson (CnChR1), which speeds up the channel closing speed, making it possible to effectively respond to light signals at least 20Hz and below. When used with Chronos, it is possible to control two different neurons with two fluorophores. Excitation light wavelength 590nm 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. The excitation wavelength is 495nm. 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.

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 frequencies) ChrimsonR: Discovered through high-throughput sequencing in Boyden's laboratory, a new type of light-controlled ion channel can respond to red light around 600nm. A point mutation of K176R was made on the basis of Chrimson (CnChR1), which speeds up the channel closing speed, making it possible to effectively respond to light signals at least 20Hz and below. When used with Chronos, it is possible to control two different neurons with two fluorophores. The excitation light wavelength is 590nm. 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.

ChrimsonR: A new type of light-controlled ion channel that can respond to red light around 600nm was discovered through high-throughput sequencing in the Boyden laboratory. A point mutation of K176R was made on the basis of Chrimson (CnChR1), which speeds up the channel closing speed, making it possible to effectively respond to light signals at least 20Hz and below. When used with Chronos, it is possible to control two different neurons with two fluorophores. The excitation light wavelength is 590nm.

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)

Virus Type

adeno-associated virus

Application

Expressing light-sensitive ion channel proteins, such as ChR2, ChrimsonR, etc., in neurons through rAAV viral vectors can specifically excite neurons and explore the effects of light stimulation on neurons, neural circuits and animal behavior.

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.