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pAAV-EF1a-double floxed-hChR2(H134R)-mCherry-WPRE-HGHpA
(Plasmid #20297)

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 20297 Plasmid sent as bacteria in agar stab 1 $65
AAV Retrograde 20297-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
  • Backbone manufacturer
    Stratagene
  • Backbone size w/o insert (bp) 5601
  • Vector type
    Mammalian Expression, AAV

Growth in Bacteria

  • Bacterial Resistance(s)
    Ampicillin
  • Growth Temperature
    37°C
  • Growth Strain(s)
    Stbl3
  • Copy number
    High Copy

Gene/Insert

  • Gene/Insert name
    channelrhodopsin-2
  • Alt name
    hChR2
  • Alt name
    Cop4
  • Alt name
    chr2
  • Species
    C. reinhardtii
  • Insert Size (bp)
    1647
  • Mutation
    humanized ChR2 gene with histidine 134 changed to arginine, to achieve higher currents
  • Tag / Fusion Protein
    • mCherry (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 n/a
  • (Common Sequencing Primers)

Resource Information

Depositor Comments

Please see http://www.optogenetics.org

for additional information.

Sequence discrepancies found during QC have no functional consequence.

Information for AAV Retrograde (Catalog # 20297-AAVrg) ( Back to top )

Purpose

Ready-to-use AAV Retrograde particles produced from pAAV-EF1a-double floxed-hChR2(H134R)-mCherry-WPRE-HGHpA (#20297). In addition to the viral particles, you will also receive purified pAAV-EF1a-double floxed-hChR2(H134R)-mCherry-WPRE-HGHpA plasmid DNA.

Humanized channelrhodopsin H134R mutant fused to mCherry, driven by the EF1a promoter. Cre-dependent. 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
  • Envelope AAV retrograde cap gene
    rAAV2-retro helper (plasmid #81070)
  • Buffer PBS + 0.001% Pluronic F-68 + 200 mM NaCl
  • Serotype AAVrg rAAV2-retro helper (plasmid #81070)
  • Purification Iodixanol gradient ultracentrifugation
  • Reporter Gene mCherry

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

Titering Method:
  • Real-time qPCR: The number of genome copies in viral preparations was measured by SYBR green real-time qPCR with primers targeting the ITR. Titer values were deduced by comparing the genomic content of the viral preparation to a standard curve of a plasmid of known concentration. Read our AAV Titration by qPCR protocol here.
Notes:
  • Purity of viral preparation: Viral preparations were subjected to polyacrylamide gel electrophoresis (PAGE) followed by silver staining and the molecular weight and relative intensity of the viral capsid proteins was analyzed. The abundance of viral capsid proteins as a fraction of total protein present in the sample was used to determine purity of the AAV preparation.
  • PCR confirmation of viral genome: PCR was carried out on the viral preparation with primers that only produce amplicons in the original (non-flipped) orientation. PCR was also carried out on the viral preparation with primers targeting the transgene. The PCR products were visualized on an agarose gel for size confirmation.
    • Transgene
      ChR2-H134R For: ATGGATTGAATCTCGCGGCA
      Cherry Rev: CTTGTACAGCTCGTCCATGCCG
    • Orientation
      EF1a For: AGTCTTGTAAATGCGGGCCA
      Cherry For: TCCGAGCGGATGTACCCCGAG
    • Orientation
      EF1a For: AGTCTTGTAAATGCGGGCCA
      Cherry Rev: CTTGTACAGCTCGTCCATGCCG

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.
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-double floxed-hChR2(H134R)-mCherry-WPRE-HGHpA was a gift from Karl Deisseroth (Addgene plasmid # 20297)