Purposehelper virus for monosynaptic tracing; to be coinjected with pAAV-TREtight-mTagBFP2-B19G
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||100798||Standard format: Plasmid sent in bacteria as agar stab||1||$65|
|AAV1||100798-AAV1||Virus (100 µL at titer ≥ 1×10¹³ vg/mL)|
This material is available to academics and nonprofits only.
Vector typeMammalian Expression, AAV
Growth in Bacteria
Growth Strain(s)NEB Stable
Copy numberHigh Copy
Gene/Insert nameTVA, EGFP, tet transactivator
Insert Size (bp)2082
- Promoter synapsin
- Cloning method Gibson Cloning
- 5′ sequencing primer unknown
- 3′ sequencing primer unknown (Common Sequencing Primers)
Information for AAV1 (Catalog # 100798-AAV1) ( Back to top )
Ready-to-use AAV1 particles produced from pAAV-syn-FLEX-splitTVA-EGFP-tTA (#100798). In addition to the viral particles, you will also receive purified pAAV-syn-FLEX-splitTVA-EGFP-tTA plasmid DNA.Helper virus for monosynaptic tracing with rabies virus; to be coinjected with pAAV-TREtight-mTagBFP2-B19G (Addgene#100799). These AAV preparations are suitable purity for injection into animals.
- 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, AAV1 cap gene
- Buffer PBS + 0.001% Pluronic F-68
- Serotype AAV1
- Purification Iodixanol gradient ultracentrifugation
- Reporter Gene EGFP
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
- 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.
Dr. Wickersham has tested these preps and he recommends the following dilution and mix:
- pAAV-syn-FLEX-splitTVA-EGFP-tTA (Addgene #100798-AAV1, lot#v15287 (1.7 × 10^13 GC∕mL): 1:200 dilution, to be mixed 50/50 by volume with diluted 100799 below:
- pAAV-TREtight-mTagBFP2-B19G (Addgene #100799-AAV1, lot#v14716 (3.2 × 10^13 GC∕mL): 1:20 dilution, to be mixed 50/50 by volume with diluted 100798 above;
When selecting what system to use, keep in mind that the two vector Addgene#100798 + Addgene# 100799 combination allows reduction of background infection by rabies virus in wild-type mice to acceptably few cells while still allowing nice amounts of transsynaptic spread in Cre+ mice (as compared to using Addgene#52473 alone). Note that EGFP can be seen only with immunostaining at these dilutions.
Dr. Wickersham recommends injecting the rabies virus 7 days after the AAVs and perfusing 7 days after that.
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-syn-FLEX-splitTVA-EGFP-tTA was a gift from Ian Wickersham (Addgene plasmid # 100798 ; http://n2t.net/addgene:100798 ; RRID:Addgene_100798)
For viral preps, please replace (Addgene plasmid # 100798) in the above sentence with: (Addgene viral prep # 100798-AAV1)
For your References section:Lhx6-positive GABA-releasing neurons of the zona incerta promote sleep. Liu K, Kim J, Kim DW, Zhang YS, Bao H, Denaxa M, Lim SA, Kim E, Liu C, Wickersham IR, Pachinis V, Hattar S, Song J, Brown SP, Blackshaw S. Nature. 2017 Aug 31;548(7669):582-587. doi: 10.1038/nature23663. Epub 2017 Aug 23. 10.1038/nature23663 PubMed 28847002