pAAV-Ef1a-DIO-PPO-Venus
(Plasmid #139505)

• Purpose: AAV vector for Cre-dependent expression of Venus-tagged parapinopsin driven by the Ef1a promoter. Used for photoswitchable control of inhibitory GPCR signaling cascades.
• Depositing Lab: Michael Bruchas
• Publication: Copits et al Neuron. 2021 Jun 2;109(11):1791-1809.e11. doi: 10.1016/j.neuron.2021.04.026. Epub 2021 May 11.

Backbone

• Vector backbone: pAAV-Ef1a-DIO-eYFP
• Backbone manufacturer: Deisseroth lab
• Backbone size w/o insert (bp): 5681
• Total vector size (bp): 7439
• 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: parapinopsin
• Alt name: PPO
• Species: Lethenteron camtschaticum
• Insert Size (bp): 1029
• GenBank ID: AB116380.1
• Promoter: Ef1a
• Tag / Fusion Protein:
  • Venus tag following 3x Ala linker (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: T7 forward primer
• 3′ sequencing primer: 5’-GGCACAGTCGAGGCGCGCCAGCGGGTTTAAACG-3’

Resource Information

• Supplemental Documents:
  • pAAV:Ef1a:DIO:PPO-Venus map
• A portion of this plasmid was derived from a plasmid made by: Akihisa Terakita, Osaka University, Japan

Terms and Licenses

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

Depositor Comments

See Koyanagi et al., PNAS 2004 (PMID: 15096614) for original cloning of lamprey parapinopsin. Please visit https://www.biorxiv.org/content/10.1101/2021.02.19.432008v1 for bioRxiv preprint.

Information for AAV8 (Catalog # 139505-AAV8)

Purpose

Ready-to-use AAV8 particles produced from pAAV-Ef1a-DIO-PPO-Venus (#139505). In addition to the viral particles, you will also receive purified pAAV-Ef1a-DIO-PPO-Venus plasmid DNA.

EF1a-driven, Cre-dependent expression of Venus-tagged parapinopsin for spatiotemporal control of inhibitory GPCR signaling cascades. 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, AAV8 cap gene
• Buffer: PBS + 0.001% Poloxamer 188
• Serotype: AAV8
• Purification: Iodixanol gradient ultracentrifugation
• Reporter Gene: Venus (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.

Information for AAV9 (Catalog # 139505-AAV9)

Purpose

Ready-to-use AAV9 particles produced from pAAV-Ef1a-DIO-PPO-Venus (#139505). In addition to the viral particles, you will also receive purified pAAV-Ef1a-DIO-PPO-Venus plasmid DNA.

EF1a-driven, Cre-dependent expression of Venus-tagged parapinopsin for spatiotemporal control of inhibitory GPCR signaling cascades. 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, AAV9 cap gene
• Buffer: PBS + 0.001% Poloxamer 188
• Serotype: AAV9
• Purification: Iodixanol gradient ultracentrifugation
• Reporter Gene: Venus (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.

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.