Syn-GCaMP6s AAV (Serotype 9)

Name: Syn-GCaMP6s AAV (Serotype 9)
SKU: AAB0062

Cat.No. : AAB0062

Titer: ≥1x10^12 GC/mL / ≥1x10^13 GC/mL Size: 30 ul/100 ul/500 ul/1 ml

Serotype: AAV Serotype 9 Storage: -80 ℃

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AAV Particle Information

Quality Control

Cat. No.	AAB0062
Description	Premade AAV particles in serotype 9 containing GCaMP6s under the control of a Syn promoter.
Serotype	AAV Serotype 9
Tag	GCaMP6s
Product Type	Adeno-associated virus particles
Biosensor	GCaMP6s-Improved SNR, slower kinetics; Green indicator
Titer	Varies lot by lot, typically ≥1x10^12 GC/mL
Size	Varies lot by lot, for example, 30 μL, 50 μL, 100 μL etc.
Storage	Store at -80℃. Avoid multiple freeze/thaw cycles.
Shipping	Frozen on dry ice

Creative Biogene ensures high-quality AAV particles by optimizing and standardizing production protocols and performing stringent quality control (QC). The specific QC experiments performed vary between AAV particle lots.
Endotoxin	Endotoxins, primarily derived from Gram-negative bacteria, can trigger adverse immune responses. Endotoxin contamination is a significant concern in the production of AAV, especially for applications in animal studies and gene therapy. Effective endotoxin quality control is essential in the development and manufacturing of AAV particles. Creative Biogene utilizes rigorous endotoxin detection methods to monitor the endotoxin level in our produced AAV particles to ensure regulatory compliance.
Purity	AAV purity is critical for ensuring the safety and efficacy of AAV-based applications.AAV capsids are composed of three main protein components, known as viral proteins: VP1, VP2, and VP3. These proteins play a critical role in the structure and functionality of the AAV capsid. Monitoring the VP1, VP2, and VP3 content in AAV preparations is essential for quality control in AAV production. Our AAV particles are tested for showing three clear bands of VP1, VP2 VP3 by SDS-PAGE.
Sterility	The AAV virus samples are inoculated into the cell culture medium for about 5 days to detect bacterial and fungal growth.
Transducibility	Upon requirement, Creative Biogene can perform in vitro or in vivo transduction assays to evaluate the ability of AAV to deliver genetic material into target cells or tissues, and assess gene expression and functional activities.
Empty vs. Full Capsids	Based-on our proprietary AAV production and purification technology, Creative Biogene can always offer AAV particles with high ratio of full capsids. If required, we can also assess the ratio for a specifc lot of AAV particles by transmission electron microscopy (TEM) or other methods.

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Gene therapy, which introduces genetic material into a patient to alter gene or protein expression, has the potential to provide a one-time cure for many currently incurable diseases. Four decades of research have shown that adeno-associated virus (AAV) appears to be the safest and most effective vector for delivering target genes to a variety of cell types for gene therapy. Inherited diseases are particularly attractive targets; these diseases are caused by genetic mutations that result in the absence or dysfunction of proteins required for cellular function. To treat such inherited diseases at the source, gene therapy can correct the disease mutation in three ways—(1) replacing the defective gene with a functional copy, (2) silencing the mutated version of the gene, and (3) adding or overexpressing a therapeutic gene or synthetic construct. Silencing can be achieved by introducing short hairpin RNA embedded in a microRNA construct or by zinc finger silencing technology. AAV can mediate both transient and permanent approaches.

AAV is a small, nonenveloped virus with a single-stranded genomic DNA of 4.7 kb flanked by 2 inverted terminal repeats (ITRs). There are 3 genes in the viral genome - Rep (replication) is responsible for viral replication and packaging, Cap (capsid) encodes 60 coat proteins that protect the genomic DNA and directly bind to the cell, and Aap (assembly activation protein) provides a scaffold for capsid assembly. Over the past few decades, recombinant AAV (rAAV) has been engineered to replace much of the viral genome with an expression cassette containing a promoter, target gene, and terminator to make it more suitable for clinical applications. Because AAVs are unable to replicate, they are a very safe vector that can drive long-term transgene expression after a single infection. In fact, the longest AAV transgene expression in primates has been reported to last for more than 15 years. The simple genome of AAV also makes it versatile and ideal for engineering.

Prolonged exposure to negative stressors can be harmful if subjects are unable to respond appropriately. In rodents, self-grooming is a frequently observed repetitive behavior believed to contribute to post-stress de-arousal with adaptive value. Here, researchers identify a rat limbic di-synaptic circuit that mediates stress-induced self-grooming with positive affective valence. This circuit links hippocampal ventral subiculum to the ventral lateral septum (LSv) and then the lateral hypothalamus tuberal nucleus. Optogenetic activation of this circuit triggers delayed but robust overgrooming in a pattern that closely resembles that induced by emotional stress. Consistently, neural activity in the LSv peaks before emotional stress-induced grooming, and inhibition of this circuit significantly suppresses emotional stress-induced grooming. These findings reveal a previously unknown limbic circuit involved in mediating stress-induced self-grooming and identify a critical role for the LSv in this ethologically important behaviour.

To understand the role of LSv in stress-mediated self-grooming behavior, researchers determined LSv neuronal population dynamics in freely moving rats in different grooming models by fiber optic photometry based on GCaMP6s reporter gene. Inject 100 nl AAV9-Syn-GCaMP6s or AAV5-hSyn-eGFP into the right LSv of rats (Figure 1a). After stress induction, neuronal calcium activity was monitored and aligned with the detected onset of grooming episodes. In both body restraint (Figure 1b) and light exposure model (Figure 1c), a significant rise in the calcium signal of LSv neurons was detected on average shortly before the onset of grooming events. Significant differences in ΔF/F were found during pre- and post-grooming periods (Figure 1b, c). In contrast, no significant changes in calcium signals were found in the swimming and spray models as well as spontaneous grooming. Furthermore, in control animals expressing eGFP in LSv neurons, there was no change in fluorescence signal during grooming behavior.

Figure 1. Activation of LSv neurons precedes emotional stress-induced grooming. (Mu M D, et al., 2020)

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