PurposeAAV-mediated expression of Jaws-KGC-GFP-ER2 under the CAG promoter, in floxed/reversed (Cre-dependent) manner.
Depositor Sequences: None.
Addgene Sequences: Full (1)
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||84445||Plasmid sent as bacteria in agar stab||1||$65|
Virus (100 µL at titer ≥ 7×10¹² vg/mL)
and Plasmid. More Information
This material is available to academics and nonprofits only.
Vector backboneAAV with CAG promter
- Backbone size w/o insert (bp) 4897
- Total vector size (bp) 6556
Vector typeMammalian Expression, AAV
Growth in Bacteria
Growth Strain(s)NEB Stable
Growth instructionsCan use Stbl3 at 30°C (use carbenicillin if using Stbl3) or DH5a at 37°C.
Copy numberLow Copy
SpeciesHaloarcula salinarum (stain Shark)
Insert Size (bp)1659
- Promoter CAG
/ Fusion Proteins
- KGC (C terminal on insert)
- GFP (C terminal on insert)
- ER2 (C terminal on insert)
- Cloning method Restriction Enzyme
- 5′ cloning site EcoRI (not destroyed)
- 3′ cloning site AscI (not destroyed)
- 5′ sequencing primer tcggcttctggcgtgtgac
- 3′ sequencing primer aaagagacagcaaccagg (Common Sequencing Primers)
The plasmid is fully sequenced in the coding sequence regions (opsin-fluorophore and important flanking regions). Multiple digestions were done to verify the vector structure. The construct and the virus were both tested in vitro.
Information for AAV Retrograde (Catalog # 84445-AAVrg) ( Back to top )
Ready-to-use AAV Retrograde particles produced from pAAV-CAG-FLEX-rc [Jaws-KGC-GFP-ER2] (#84445). In addition to the viral particles, you will also receive purified pAAV-CAG-FLEX-rc [Jaws-KGC-GFP-ER2] plasmid DNA.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
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
Viral Quality Control
- 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.
- 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.
- In-vivo expression: AAVrg-CAG-FLEX-rc [Jaws-KGC-GFP-ER2] particles were injected into brain regions of an RBP4-Cre mouse. GFP expression was visualized two weeks later by direct fluorescence and exhibits retrograde transport from the injection site. You can view the in-vivo expression and details here or in the image section at the top of this page.
- 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.
Jaws Rev: CTGATCTGGGTGGCCACAAT
37825 Rev: GATATAGACGTTGTGGCTGTTGTAGTTG
WPRE Rev: GCAGAATCCAGGTGGCAACA
Jaws For: AGCAGAAAGGCGAACACGTA
WPRE Rev: GCAGAATCCAGGTGGCAACA
Jaws Rev: CTGATCTGGGTGGCCACAAT
- Next-generation sequencing of viral genome: Next-generation sequencing was performed on viral genomes that were isolated from the final viral preparation. Sequencing results were analyzed to confirm the identity and integrity of the viral genome and the absence of unexpected DNA contaminants.
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 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 0.01-0.03% of viral vectors in our typical production protocol. This can lead to a small number of cells exhibiting Cre-independent transgene expression in vivo. To address this, we recommend titrating to find the optimal AAV dosage required for Cre-dependent transgene expression and function in vivo. This may include reducing the viral vector dosage in order to reduce the likelihood of Cre-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-CAG-FLEX-rc [Jaws-KGC-GFP-ER2] was a gift from Edward Boyden (Addgene plasmid # 84445)
For viral preps, please replace (Addgene plasmid # 84445) in the above sentence with: (Addgene viral prep # 84445-AAVrg)
For your References section:Noninvasive optical inhibition with a red-shifted microbial rhodopsin. Chuong AS, Miri ML, Busskamp V, Matthews GA, Acker LC, Sorensen AT, Young A, Klapoetke NC, Henninger MA, Kodandaramaiah SB, Ogawa M, Ramanlal SB, Bandler RC, Allen BD, Forest CR, Chow BY, Han X, Lin Y, Tye KM, Roska B, Cardin JA, Boyden ES. Nat Neurosci. 2014 Aug;17(8):1123-9. doi: 10.1038/nn.3752. Epub 2014 Jul 6. 10.1038/nn.3752 PubMed 24997763