AAV-EF1a-DIO-GCaMP6f-P2A-nls-dTomato
(Plasmid #51083)

• Purpose: Cre dependent expression of GCaMP6f Ca sensor and physically separate nuclear localized dTomato fluorophore
• Depositing Lab: Jonathan Ting
• Publication: Ting GCaMP6 plasmids (unpublished)

Backbone

• Vector backbone: pAAV-EF1a-DIO-WPRE-BGHpA
• Backbone manufacturer: custom
• Backbone size w/o insert (bp): 5320
• Total vector size (bp): 7484
• Modifications to backbone: Altered cloning sites and replaced HGHpA with smaller BGHpA
• 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: GCaMP6f
• Alt name: G6f
• Alt name: GCaMP6 fast
• Species: Synthetic
• Insert Size (bp): 2160
• Promoter: hEF1a
• Tag / Fusion Protein:
  • P2A-nls-dTomato (C terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: AscI (not destroyed)
• 3′ cloning site: SfoI (destroyed during cloning)
• 5′ sequencing primer: gccagcttggcacttgatgtaattctcc
• 3′ sequencing primer: CCATACGGGAAGCAATAGCATG

Resource Information

• A portion of this plasmid was derived from a plasmid made by: The GCaMP6f portion was derived from Addgene plasmid #40755 (Douglas Kim, Janelia Farms).
• Articles Citing this Plasmid:
  • 5 References

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Takara Bio Limited Use Label License (formerly Clontech)
• Industry Terms:
  • Not Available to Industry

Depositor Comments

The addition of the P2A-nls-dTomato allows for easy identification of GCaMP6 expressing cells exhibiting nuclear red fluorescence that does not significantly overlap with the cytosolic GCaMP signal. The GCaMP and fluorophore are physically uncoupled.

Permissions were obtained from Douglas Kim and the Clontech licensing office for depositing this new plasmid.

Information for AAV Retrograde (Catalog # 51083-AAVrg)

Purpose

Ready-to-use AAV Retrograde particles produced from AAV-EF1a-DIO-GCaMP6f-P2A-nls-dTomato (#51083). In addition to the viral particles, you will also receive purified AAV-EF1a-DIO-GCaMP6f-P2A-nls-dTomato plasmid DNA.

Cre-dependent GCaMP6f calcium sensor and physically separate nuclear tdTomato, under control of the EF1a promoter. These AAV were produced with a retrograde serotype, which permits retrograde access to projection neurons. These AAV preparations are suitable purity for injection into animals.

Delivery

• Volume: 100 µL
• Titer: ≥5×10¹² 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, AAV retrograde cap gene from rAAV2-retro helper (plasmid #81070)
• Buffer: PBS + 0.001% Poloxamer 188 + 200 mM NaCl
• Serotype: AAV retrograde (AAVrg)
• Purification: Iodixanol gradient ultracentrifugation
• Reporter Gene: nuclear dTomato (physically separate, not a fusion protein)

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

Retrograde functionality is dependent on high viral titers. Addgene recommends not diluting your AAV preps prior to use.

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.