PurposeFlp-dependent hM4D(Gi) DREADD-mCherry
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||154867||Standard format: Plasmid sent in bacteria as agar stab||1||$75|
|AAV Retrograde||154867-AAVrg||Virus (100 µL at titer ≥ 7×10¹² vg/mL) and Plasmid.|
This material is available to academics and nonprofits only.
Backbone manufacturerGether lab - modified from Addgene plasmid #55641
- Backbone size w/o insert (bp) 4824
- Total vector size (bp) 6993
Vector typeMammalian Expression, AAV ; FLP-FRT
Growth in Bacteria
Growth Strain(s)NEB Stable
Copy numberHigh Copy
Insert Size (bp)2169
- Promoter Human Synapsin-1
/ Fusion Protein
- mCherry (C terminal on insert)
- Cloning method Restriction Enzyme
- 5′ cloning site Asc1 (not destroyed)
- 3′ cloning site Nhe1 (not destroyed)
- 5′ sequencing primer actcagcgctgcctcagtct
- 3′ sequencing primer gatacaaaggcattaaagcagcg (Common Sequencing Primers)
Terms and Licenses
- Not Available to Industry
Information for AAV Retrograde (Catalog # 154867-AAVrg) ( Back to top )
Ready-to-use AAV Retrograde particles produced from pAAV-hSyn-fDIO-hM4D(Gi)-mCherry-WPREpA (#154867). In addition to the viral particles, you will also receive purified pAAV-hSyn-fDIO-hM4D(Gi)-mCherry-WPREpA plasmid DNA.Syn-driven, Flp-dependent hM4D(Gi) receptor with an mCherry reporter for CNO-induced neuronal inhibition. 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.
- 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
- Reporter Gene mCherry (Flp-dependent)
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
- Not Available to Industry
Viral 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 CommentsRetrograde 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 0.1-0.8% of viral particles in our typical production protocol. This can lead to a small number of cells exhibiting Flp-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 Flp-dependent transgene expression and function in vivo. This may include reducing the viral particle dosage in order to reduce the likelihood of Flp-independent expression.
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-hSyn-fDIO-hM4D(Gi)-mCherry-WPREpA was a gift from Ulrik Gether (Addgene plasmid # 154867 ; http://n2t.net/addgene:154867 ; RRID:Addgene_154867)
For viral preps, please replace (Addgene plasmid # 154867) in the above sentence with: (Addgene viral prep # 154867-AAVrg)
For your References section:Locomotor- and Reward-Enhancing Effects of Cocaine Are Differentially Regulated by Chemogenetic Stimulation of Gi-Signaling in Dopaminergic Neurons. Runegaard AH, Sorensen AT, Fitzpatrick CM, Jorgensen SH, Petersen AV, Hansen NW, Weikop P, Andreasen JT, Mikkelsen JD, Perrier JF, Woldbye D, Rickhag M, Wortwein G, Gether U. eNeuro. 2018 Jun 18;5(3). pii: eN-NWR-0345-17. doi: 10.1523/ENEURO.0345-17.2018. eCollection 2018 May-Jun. 10.1523/ENEURO.0345-17.2018 PubMed 29938215