pAAV-CAG-Flex-mRuby2-GSG-P2A-GCaMP6s-WPRE-pA
(Plasmid #68717)

• Purpose: Cre-dependent bicistronic vector expressing mRuby2 and GCaMP6s from a single open reading frame.
• Depositing Labs: Tobias Bonhoeffer, Mark Huebener, Tobias Rose
• Publication: Rose et al Science. 2016 Jun 10;352(6291):1319-22. doi: 10.1126/science.aad3358.

Backbone

• Vector backbone: pAAV
• Total vector size (bp): 7112
• Vector type: AAV

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): Stbl3
• Growth instructions: Due to ease of recombination, AAV and lentivirus vectors should be amplified in a recombination deficient bacteria strain such as Invitrogen's OneShot Stbl3 cells. Check for integrety of ITR sites with SmaI digest.
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: mRuby2-P2A-GCaMP6s
• Species: R. norvegicus (rat), G. gallus (chicken); A. victoria (jellyfish)
• Insert Size (bp): 2124
• Mutation: Cre-dependent expression from inverted open reading frame (i.e. 'FLEXED')
• Promoter: CAG-FLEX

Cloning Information

• Cloning method: Unknown
• 5′ sequencing primer: actcagcgctgcctcagtct
• 3′ sequencing primer: gtttgtacaaatgatgacagcgaag

Resource Information

• Articles Citing this Plasmid:
  • 2 References

Terms and Licenses

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

Depositor Comments

mRuby2:
Article: Improving FRET dynamic range with bright green and red fluorescent proteins. Lam et al (Nat Methods. 2012 Sep 9. doi: 10.1038/nmeth.2171. PubMed)
Addgene Plasmid 40260

GCaMP6s:
Article: Ultrasensitive fluorescent proteins for imaging neuronal activity. Chen et al (Nature. 2013 Jul 18;499(7458):295-300. doi: 10.1038/nature12354. PubMed)
Addgene Plasmid 40753

Information for AAV1 (Catalog # 68717-AAV1)

Purpose

Ready-to-use AAV1 particles produced from pAAV-CAG-Flex-mRuby2-GSG-P2A-GCaMP6s-WPRE-pA (#68717). In addition to the viral particles, you will also receive purified pAAV-CAG-Flex-mRuby2-GSG-P2A-GCaMP6s-WPRE-pA plasmid DNA.

CAG-driven, Cre dependent, GCaMP6s calcium sensor and bicistronic, physically separate mRuby2 expression. 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, AAV1 cap gene
• Buffer: PBS + 0.001% Poloxamer 188
• Serotype: AAV1
• Purification: Iodixanol gradient ultracentrifugation
• Reporter Gene: mRuby2 (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:
  • 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.

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.