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pAAV-EF1a-fDIO-Cre
(Plasmid #121675)

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 121675 Standard format: Plasmid sent in bacteria as agar stab 1 $65
AAV9 121675-AAV9 Virus (100 µL at titer ≥ 1×10¹³ vg/mL) and Plasmid. More Information
$380
AAV Retrograde 121675-AAVrg Virus (100 µL at titer ≥ 7×10¹² vg/mL) and Plasmid. More Information
$380

This material is available to academics and nonprofits only.

Backbone

  • Vector backbone
    pAAV-Ef1a-fDIO EYFP (Addgene #55641)
  • Backbone manufacturer
    Karl Deisseroth
  • Backbone size w/o insert (bp) 6323
  • Total vector size (bp) 6746
  • Modifications to backbone
    Replaced EYFP with NLS-CRE-HA
  • Vector type
    Mammalian Expression, AAV

Growth in Bacteria

  • Bacterial Resistance(s)
    Ampicillin
  • Growth Temperature
    37°C
  • Growth Strain(s)
    NEB Stable
  • Copy number
    High Copy

Gene/Insert

  • Gene/Insert name
    NLS-CRE-HA
  • Species
    Synthetic
  • Insert Size (bp)
    1047
  • Promoter EF1a
  • Tags / Fusion Proteins
    • SV40-NLS (N terminal on insert)
    • 3xHA (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 TCAAGCCTCAGACAGTGGTTC
  • 3′ sequencing primer CATAGCGTAAAAGGAGCAACA
  • (Common Sequencing Primers)

Resource Information

  • Terms and Licenses

Information for AAV9 (Catalog # 121675-AAV9) ( Back to top )

Purpose

Ready-to-use AAV9 particles produced from pAAV-EF1a-fDIO-Cre (#121675). In addition to the viral particles, you will also receive purified pAAV-EF1a-fDIO-Cre plasmid DNA.

EF1a-driven, Flp recombinase dependent expression of Cre recombinase. These AAV preparations are suitable purity for injection into animals.

Delivery

  • Volume 100 µL
  • Titer ≥ 1×10¹³ vg/mL
  • Pricing $350 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% Pluronic F-68
  • Serotype AAV9
  • 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

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 recombinase-dependent vectors in vivo: FRT sites in fDIO plasmids are known to recombine during DNA amplification and viral vector production, which may result in a minority of Flp-activated (i.e., "flipped") viral vectors. Addgene has measured this occurs in 0.01-0.03% of viral vectors in our typical production protocol. This can lead to a small number of cells exhibiting Flp-independent transgene expression in vivo. To address this, we recommend titrating to find the optimal AAV dosage required for Flp-dependent transgene expression and function in vivo. This may include reducing the viral vector dosage in order to reduce the likelihood of Flp-independent expression.

Information for AAV Retrograde (Catalog # 121675-AAVrg) ( Back to top )

Purpose

Ready-to-use AAV Retrograde particles produced from pAAV-EF1a-fDIO-Cre (#121675). In addition to the viral particles, you will also receive purified pAAV-EF1a-fDIO-Cre plasmid DNA.

EF1a-driven, Flp recombinase dependent expression of Cre recombinase. 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 ≥ 7×10¹² vg/mL
  • Pricing $350 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% Pluronic F-68 + 200 mM NaCl
  • Serotype AAV retrograde (AAVrg)
  • 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

Resource Information

Viral Quality Control

b>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 recombinase-dependent vectors in vivo: FRT sites in fDIO plasmids are known to recombine during DNA amplification and viral vector production, which may result in a minority of Flp-activated (i.e., "flipped") viral vectors. Addgene has measured this occurs in 0.01-0.03% of viral vectors in our typical production protocol. This can lead to a small number of cells exhibiting Flp-independent transgene expression in vivo. To address this, we recommend titrating to find the optimal AAV dosage required for Flp-dependent transgene expression and function in vivo. This may include reducing the viral vector dosage in order to reduce the likelihood of Flp-independent expression.

How to cite this plasmid ( Back to top)

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.

  • For your Materials & Methods section:

    pAAV-EF1a-fDIO-Cre was a gift from Esteban Engel & Alexander Nectow (Addgene plasmid # 121675 ; http://n2t.net/addgene:121675 ; RRID:Addgene_121675)

    For viral preps, please replace (Addgene plasmid # 121675) in the above sentence with: (Addgene viral prep # 121675-AAV9) or (Addgene viral prep # 121675-AAVrg)

  • For your References section:

    Regulation of Energy Expenditure by Brainstem GABA Neurons. Schneeberger M, Parolari L, Das Banerjee T, Bhave V, Wang P, Patel B, Topilko T, Wu Z, Choi CHJ, Yu X, Pellegrino K, Engel EA, Cohen P, Renier N, Friedman JM, Nectow AR. Cell. 2019 Jun 24. pii: S0092-8674(19)30619-1. doi: 10.1016/j.cell.2019.05.048. 10.1016/j.cell.2019.05.048 PubMed 31257028