Vigene Biosciences
Marketing and Sales Manager
Genetically encoded fluorescent biosensors are powerful tools used to analyze biomolecular interactions in optogenetics. Biosensors have been hailed as a solution to deciphering cell signaling, particularly labeling of active neural circuits in vivo. Structurally, they comprise a fluorescent protein (FP) attached to an additional protein sequence, for sensitivity to small biomolecules like calcium ions (Ca2+). The small structure of biosensors makes them ideal for introduction into cells, tissues or organisms and detection using fluorescence microscopy. One of their key advantages is long-term imaging, paving an exciting opportunity to view neuronal activities. In this article, we discuss some key features of adeno-associated virus (AAV) Biosensors, providing a brief introduction to biosensors and AAV, followed by how these tools help researchers gain insight into events related to well-known diseases (such as visualizing neuronal electrical potentials in neurological disease models).
AAV Biosensors
Biosensors such as genetically encoded calcium indicators (GECIs) report Ca2+ changes with the added advantage of noninvasive imaging of defined cells and compartments. With Ca2+ detection, fusion of a FP like GFP to a specific binding domain can be used to report on the production of certain signaling molecules. Ca2+ sensors consist of a molecular switch containing calmodulin (CaM), and M13 (a synthetic peptide from myosin light chain kinase) inserted into a circularly permuted GFP in which the native N- and C-termini are linked together; new termini are generated from within the core beta-barrel structure. Addition of Ca2+ causes CaM to bind M13, leading to increased GFP fluorescence and providing a means to probe the biochemical function and dynamics through sensitive changes in fluorescence properties.
AAV viral transduction system
Viral vectors allow temporal and spatial controlled expression of genes of interest (GOI) in vitro and in vivo. One such viral vector, the recombinant adeno-associated virus (rAAV) has become a staple for gene delivery in different tissues and organs, including brain.
AAV is a single-stranded DNA virus with a small (approximately 20-nm) protein capsule unable to replicate in the absence of helpervirus co-infections, typically adenovirus or herpesvirus. Infection produces mild immune responses considered nonpathogenic; this allows for low biosafety-level requirements when working with rAAV compared with other viruses. rAAV vector genomes persist as episomal concatemers, limiting the risk of insertional mutagenesis and making them preferred over lentiviruses, which integrate into the genome, in optogenetics experiments. Because of their low immunogenicity and the absence of cytotoxic responses, AAV-based expression systems offer the opportunity to express GOI over an extended time frame (e.g., months) in quiescent cells. Furthermore, AAV serotypes (AAV1-10) that differ in tropism, or the types of cells they infect, make AAV an extremely useful system for preferentially transducing certain cell types, e.g., neurons, heart and kidney.
The goal of fluorescence-based biosensors packaged into AAV is use in live-cell imaging: converting a molecular event into an optical signal that can be detected by fluorescence microscopy.
Choosing an appropriate biosensor
AAV Biosensors perfectly monitor and measure fluctuations in molecular signals in vitro or in vivo. Herein we briefly describe key single-wavelength AAV Biosensors—those monitoring calcium and glutamate and their primary application(s).
Calcium
Calcium is an important regulator of cellular signaling events, with calcium indicators having extensive use for imaging and measuring changes in Ca2+ associated with neural activity. The following GECI biosensors offer rapid response to Ca2+ changes across research needs.
CaMPARI, or Calcium Modulated Photoactivatable Ratiometric Integrator, is a photoconvertible protein construct enabling imaging of the integrated calcium activity of large cell populations over defined time windows. This biosensor is suitable for labeling of “active” cells within a tissue (such as brain) during stimulus or behavior in model organisms.
GCaMP sensors comprise a family of ultrasensitive green fluorescent indicator proteins that facilitate measurement of synaptic calcium signals. Three common types are: GCaMP3, GCaMP5 and GCaMP6. GCaMP3 reliably detects three or more action potentials in short bursts in several systems in vivo; GCaMP5 provides a more reliable measure of sensitive detection of neural activity in vivo; GCaMP6 offers improved engineering, allowing for increased signal-to-noise ratio and faster kinetics. Each sensor has improved properties relative to parent sensor GCaMP3 and is suitable for applications studying high-frequency neuronal activity.
RCaMP (jRCaMP1a and jRCaMP1b) is similar to GCaMP, but because it is generated from a fusion of red fluorescent protein (RFP), it provides a new color channel for single-wavelength functional imaging. RCaMP is predominantly used in multifunctional imaging for drug screening and for imaging cellular activity in cells and organisms already expressing GFP.
jRCEGO1a and jRCEGO1b, similarly to GCaMP and RCaMP, have been generated from a fusion of the mApple-based (green) fluorescent protein. These have strong applications focused on in vivo imaging, specifically deep tissue imaging.
Glutamate
Glutamate, the major neurotransmitter in the brain, plays a critical role in nearly all aspects of normal brain function. Dysregulation is associated with stroke and neurodegenerative disorders, including Alzheimer's disease.
iGluSnFR delivers an improved means of directly mapping excitatory synaptic activity. It offers applications that complement existing imaging methods for neural activity and signaling events; glutamate imaging studies in nonneuronal tissues can also benefit from iGluSnFR.
Conclusion
AAV Biosensors provide a snapshot of intracellular interactions, specifically active brain circuitry. Using fluorescent protein-based AAV Biosensors allows permanent marking of active cells over short time scales, providing a window into the real-time biochemistry of living cells and whole organisms.
Vigene Biosciences offers ready-to-transduce AAV Biosensors of the above mentioned types. Vigene's AAV Biosensor products come ready to use, with a choice of promoter and the ability to include Cre inducible (FLEx-ON) expression. Vigene’s AAV Biosensors are made possible through a worldwide license* from Janelia Research Campus, HHMI. With a ready-to-inject format and overnight delivery, these products facilitate sophisticated in vivo imaging experiments.
Also available for immediate download: the AAV Biosensors Handbook and Product Information.