pAAV-CAG-DIO-ChR2(H134R)-eYFP
(Plasmid #127090)

• Purpose: Cre-dependent AAV expression of humanized ChR2 (with H134R mutation) fused to eYFP for optogenetic activation
• Depositing Lab: Viviana Gradinaru
• Publication: Rajendran et al Nat Commun. 2019 Apr 26;10(1):1944. doi: 10.1038/s41467-019-09770-1.

Backbone

• Vector backbone: pAAV
• Vector type: Mammalian Expression, AAV

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): NEB Stable
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: Channelrhodopsin-2
• Alt name: ChR2
• Alt name: hChR2(H134R)
• Species: C. reinhardtii
• Insert Size (bp): 1662
• Mutation: Humanized ChR2 gene with histidine 134 changed to arginine (H134R), to achieve higher currents
• Promoter: CAG
• Tag / Fusion Protein:
  • eYFP (C terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: MluI (not destroyed)
• 3′ cloning site: KpnI (not destroyed)
• 5′ sequencing primer: n/a
• 3′ sequencing primer: n/a

Resource Information

• Supplemental Documents:
  • pAAV-CAG-DIO-ChR2(H134R)-eYFP.gbk
• A portion of this plasmid was derived from a plasmid made by: Karl Deisseroth* (see below)
• Articles Citing this Plasmid:
  • 2 References

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

*Note: This construct is based on Addgene ID 20298 (the Ef1a promoter was changed to the CAG promoter).

Information for AAV PHP.eB (Catalog # 127090-PHPeB)

Purpose

Ready-to-use AAV PHP.eB particles produced from pAAV-CAG-DIO-ChR2(H134R)-eYFP (#127090). In addition to the viral particles, you will also receive purified pAAV-CAG-DIO-ChR2(H134R)-eYFP plasmid DNA.

CAG-driven, Cre-dependent expression of humanized channelrhodopsin H134R mutant fused to EYFP, for optogenetic activation. These AAV were produced with the PHPeB serotype, which permits efficient transduction of the central nervous system. These AAV preparations are suitable purity for injection into animals.

Delivery

• Volume: 100 µL
• Titer: ≥ 1×10¹³ vg/mL
• Pricing: $375 USD for preparation of 100 µL virus + $30 USD for plasmid.
• Storage: Store at -80℃. Thaw just before use and keep on ice.
• Shipment: Viral particles are shipped frozen on dry ice. Plasmid DNA (≥ 200ng) will also be included in the shipment.

Viral Production & Use

• Packaging Plasmids: encode adenoviral helper sequences and AAV rep gene, PHP.eB cap gene
  pUCmini-iCAP-PHP.eB (plasmid #103005)
• Buffer: PBS + 0.001% Poloxamer 188 + 200 mM NaCl
• Serotype: PHPeB (plasmid #103005)
• Purification: Iodixanol gradient ultracentrifugation
• Reporter Gene: EYFP (Cre-dependent)

Biosafety

Requestor is responsible for compliance with their institution's biosafety regulations. Lentivirus is generally considered BSL-2. AAV is generally considered BSL-1, but may require BSL-2 handling depending on the insert. Biosafety Guide

Terms and Licenses

• Academic/Nonprofit Terms:
  • Ancillary Agreement for Penn Vectors
  • Terms of Use for Viral Vectors
• Industry Terms:
  • Not Available to Industry

Viral Quality Control

Quality Control:

• Addgene ensures high quality viral vectors by optimizing and standardizing production protocols and performing rigorous quality control (QC) (see a list of our QC assays). The specific QC assays performed varies for each viral lot. To learn which specific QC assays were performed on your lot, please contact us.
• Titer: the exact titer of your sample will be reported on the tube. The titer you see listed on this page is the guaranteed minimum titer. See how titers are measured.

Please note that this vector may have a higher level of recombination than our other FLEX vectors. We are making it available because it is still useful to some scientists, but understand that you may see expression in the absence of Cre.

Visit our viral production page for more information.

Addgene Comments

Using FLEX vectors in vivo: LoxP sites in FLEX plasmids are known to recombine during DNA amplification and viral vector production, which may result in a minority of Cre-activated (i.e., "flipped") viral vectors. Addgene has measured this occurs in 0.1-0.8% of viral particles in our typical production protocol. This can lead to a small number of cells exhibiting Cre-independent transgene expression in vivo. To address this, it is necessary to optimize the injection volume and viral titer to find the optimal AAV dosage required for Cre-dependent transgene expression and function in vivo. This may include reducing the viral particle dosage in order to reduce the likelihood of Cre-independent expression.

Citation Information: When using the PHP.eB serotype in future publications, please acknowledge Viviana Gradinaru and cite Chan et al., Nat Neurosci, 20(8):1172-1179. Pubmed.