pAAV-EF1a-mitoGFP-DIO
(Plasmid #174112)

• Purpose: Cre-dependent expression of mitochondria-targeted Green Fluorescent Protein under control of the EF1a promoter
• Depositing Lab: Maarten Kole
• Publication: Kole et al Nat Commun. 2022 Dec 9;13(1):7598. doi: 10.1038/s41467-022-35350-x.

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• Serotype: Select serotype for details See details about
• Pricing: Select serotype and quantity $ USD for preparation of µL virus + $32 USD for plasmid.
• How this works:
  • Place a request for a quantity of 2 (0.2 mL), 10 (1 mL), 25 (2.5 mL), or 50 (5 mL). Our all-inclusive pricing includes DNA production and QC.
  • Addgene will quickly confirm that we can produce a high-quality prep for you.
  • Track your request and place an order from within your account. Payment information must be added before we can begin processing your order.
  • Receive your prep in 6–9 weeks after the MTA is approved by your organization.
  • Learn more about our Packaged on Request Service.

Backbone

• Vector backbone: pAAV-EF1a-double floxed-mCherry-WPRE-HGHpA
• Backbone manufacturer: Karl Deisseroth
• Backbone size w/o insert (bp): 5604
• Total vector size (bp): 6476
• Vector type: 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: mitoGFP
• Alt name: Cox8
• Species: H. sapiens (human)
• Insert Size (bp): 872
• Entrez Gene: COX8A (a.k.a. COX, COX8, COX8-2, COX8L, MC4DN15, VIII, VIII-L)
• Promoter: Ef1a
• Tags / Fusion Proteins:
  • Cox8 targeting sequence (N terminal on insert)
  • GFP (C terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: AscI (not destroyed)
• 3′ cloning site: NheI (not destroyed)
• 5′ sequencing primer: GGATTGTAGCTGCTATTAGC
• 3′ sequencing primer: GGCAAATAACTTCGTATAGGA

Resource Information

• A portion of this plasmid was derived from a plasmid made by: The mitoGFP gene was derived from plasmid #44385, deposited by the lab of Pantelis Tsoulfas
• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

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

Depositor Comments

Please visit https://doi.org/10.1101/2022.04.07.487496 for bioRxiv preprint.

Information for AAV2 (Catalog # 174112-AAV2)

Purpose

Ready-to-use AAV2 particles produced from pAAV-EF1a-mitoGFP-DIO (#174112). In addition to the viral particles, you will also receive purified pAAV-EF1a-mitoGFP-DIO plasmid DNA.

EF1a-driven, Cre-dependent expression of mitochondria-targeted GFP. These AAV preparations are suitable purity for injection into animals.

Delivery

• Volume: 100 µL
• Titer: ≥ 7×10¹² vg/mL
• Pricing: $1000 USD for preparation of 100 µL virus + $32 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, AAV2 cap gene
• Buffer: PBS + 0.001% Pluronic F-68 + 200 mM NaCl
• Serotype: AAV2
• Purification: Iodixanol gradient ultracentrifugation

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.