Ready to use AAV Biosensors
Adeno-associated virus (AAV) Biosensor products come as ready-to-use AAV virus. The viruses encode your chosen biosensor, either calcium or glutamate sensor and are ready for in vivo injection.
Biosensors are genetically engineered fluorescent proteins (FP) attached to an additional protein sequence which makes them sensitive to small biomolecules or other physiological intracellular processes (e.g. Ca2+). These biosensors are introduced to cells, tissues or organisms to detect changes by fluorescent microscopy or spectrum changes. Many biosensors permit long-term imaging and can be engineered to specifically target cellular compartments or organelles. Additionally, biosensors permit signaling pathway exploration or allow the measurement of a biomolecule. They do all this while preserving both spatial and temporal cellular processes.
AMSBIO offers a range of Calcium or Glutamate Biosensor products that come ready-to-use, with a choice of promoter and the ability to include the Cre inducible (FLEx-ON) expression. Vigene have packaged these indicators into the most commonly used AAV serotypes (AAV8 and AAV9). Should you require a different serotype, please contact us.
Fig 1.Different types of FP-based biosensor. The fusion of an FP such as GFP to a specific binding domain (grey) can be used to report on the production of certain signaling molecules. Ca2+ sensors consists of a molecular switch that contains calmodulin (CaM) and M13 inserted into a circularly permuted GFP, in which the native N- and C-termini of GFP are linked together, and new termini are generated from within the core b-barrel structure of GFP; the addition of Ca2+ causes CaM to bind to M13, which leads to increased GFP fluorescence.
CaMPARI calcium indicators
CaMPARI, or Calcium Modulated Photoactivatable Ratiometric Integrator is a photoconvertible protein construct, enables imaging of integrated calcium activity of large populations of cells over a defined time window. Calcium indicators have found extensive use for imaging and measuring changes in Ca2+ levels associated with neural activity.
CaMPARI is based on EosFP, a fluorescent protein whose emission changes from green to red upon irradiation with UV-light (~400 nm). By engineering libraries of EosFP variants, researchers were able to develop a protein that undergoes significantly faster green-to-red photoconversion (PC) only when calcium is present while PC light is applied. This permanent conversion provides the ability to record calcium activity for all areas illuminated by PC-light. The red fluorescence intensity correlates with calcium activity.
Key advantage of CaMPARI
• Image total calcium activity during defined time windows (gated by photoconversion light).
• Not restricted to the field of view of a microscope, as during real-time calcium imaging with e.g. GCaMP.
• Enables higher-throughput calcium assays with cultured cells.
• Calcium activity imaging across large cell populations and/or tissues.
• Labeling of “active” cells within a tissue (such as brain) during stimulus or behavior in model organisms.
• Tracing of neurons based on their calcium activity level.
GCaMP calcium indicators
GCaMP is a genetically encoded calcium indicator (GECI) which was generated from a fusion of the green fluorescent protein (GFP), calmodulin, and M13, a peptide sequence from myosin light chain kinase. Upon binding of GECI to Ca2+ there is an induction of a change in fluorescence signal. It is this change in signal that allows for measurement of the action potentials, and other receptor activation events, that trigger Ca2+ fluxes.
Key advantage of GCaMP
• Genetically specified for Ready for use studies in vivo.
• GCaMP6 are the 6th generation of GCaMP, offering improved engineering for increased signal-to-noise ratio and much faster kinetics, when compared to previous versions, specifically, GCaMP3 and GCaMP5G.
• GCaMP3 is the 3rd generation of GCaMP and reliably detects three or more action potentials in short bursts in several systems in vivo
RCaMP calcium indicators
RCaMP is a genetically encoded calcium indicator (GECI) which was generated from a fusion of the red fluorescent protein (RFP), calmodulin, and M13, a peptide sequence from myosin light chain kinase. Upon the binding of GECI to Ca2+ there is an induction of a change in fluorescence signal. It is this change in signal which allows for measuring the action potentials, and other receptor activation events, that trigger Ca2+ fluxes.
Key advantage of RCaMP6:
• Provides new color channels for single-wavelength functional imaging.
• Minimal bleed through into green channel, unlike other biosensors.
• Compatible with optogenetic activation/silencing via tools such as channel rhodopsin-2, facilitating “read/write” optogenetics.
• Tuneable affinity under control of various promoters.
• Detection using standard optical and nano technologies.
jRGECO1 calcium indicators
jRGECO1 is a genetically encoded calcium indicator (GECI) which was generated from a fusion of the mApple-based (green) fluorescent protein (FP), calmodulin, and M13, a peptide sequence from myosin light chain kinase. Upon the binding of GECI to Ca2+ there is an induction of a change in fluorescence signal. It is this change in signal which allows for measuring the action potentials, and other receptor activation events, that trigger Ca2+ fluxes.
Key advantage of jRGECO1
• Increased red fluorescence sensitivity, comparable to GCaMP6.
• Best performer for tracking sensitive stimuli; has advantages in response speed.
• Facilitate deep-tissue imaging, dual-color imaging together with GFP-based reporters, and the use of optogenetics in combination with calcium imaging.
iGluSnFR Glutamate indicator
Glutamate, an important signaling molecule, is the major neurotransmitter in the brain, playing a critical role in nearly all aspects of normal brain function. Dysregulation in glutamate is associated with stroke and neurodegenerative disorders including Alzheimer's disease.
Recently developed glutamate optical sensors are composed of glutamate-binding proteins that are coupled to fluorescent read-outs. The new and improved iGluSnFR has increased intensity and is constructed from E. coli GltI and circularly permutated GFP. Additionally, an optimized single wavelength glutamate sensor has been engineered in vitro for maximum fluorescence response. Given that this sensor is much brighter than existing options, has a rapid response time it can be used for two-color imaging experiments and long-term in vivo imaging of glutamate signaling in worms, zebrafish, and mice.
The iGluSnFR construct delivers improved means to directly map excitatory synaptic activity in the brain. This construct will complement existing imaging methods for studies of neural activity and signaling events. Glutamate imaging studies in non-neuronal tissues will also benefit from the improved performance of iGluSnFR.
Key advantage of iGluSnFR
• Extremely rapid glutamate detection with high spatial resolution.
• Improved signal-to-noise ratio compared to existing fluorescent glutamate biosensors.
• Genetically-encoded, thereby can be targeted to specific cellular populations and sub-cellular locations.
• Enables direct visualization of synaptic release (as opposed to Ca2+ imaging).
• Long-term in vivo imaging of glutamate signaling in worms, zebrafish, and mice.
Rapid detection of glutamate, improved SNR; direct visualization of synaptic release (as opposed to Ca2+ imaging)
Legal Statement of AAV Biosensor:
1. AAV Biosensors are covered under US Patents #14/350,199; #8,629,256, #14/800,814, #14/800,814, #14/941,406, #14/974,483, 14/941,406, 14/974,483, 14/452,428 and foreign equivalents and licensed from Janelia Research Campus, HHMI, Janelia, Virginia, USA.
2. The products and the reagents generated from these services shall be used as tools for research purposes, and shall exclude any human or clinical use.
3. The purchase of the AAV Biosensor Products coveys to the purchaser the limited, non-transferable right to use the products purchased and the reagents generated from Vigene Biosciences Inc. services and any related material solely for Research Purposes only, not for any Commercial Purposes.