pAAV-TREtight-mTagBFP2-B19G
(Plasmid #100799)

• Purpose: helper virus for monosynaptic tracing; to be coinjected with pAAV-syn-FLEX-splitTVA-EGFP-tTA
• Depositing Lab: Ian Wickersham
• Publication: Liu et al Nature. 2017 Aug 31;548(7669):582-587. doi: 10.1038/nature23663. Epub 2017 Aug 23.

Backbone

• Vector backbone: pAAV
• Backbone manufacturer: Stratagene
• Vector type: Mammalian Expression, AAV

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: mTagBFP2 and rabies virus SAD B19 strain glycoprotein genes (B19G)
• Species: Synthetic
• Insert Size (bp): 2337
• Promoter: TREtight

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: unknown
• 3′ sequencing primer: unknown

Resource Information

• Articles Citing this Plasmid:
  • 9 References

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Evrogen Limited Use Label License for FPs
• Industry Terms:
  • Not Available to Industry

Information for AAV1 (Catalog # 100799-AAV1)

Purpose

Ready-to-use AAV1 particles produced from pAAV-TREtight-mTagBFP2-B19G (#100799). In addition to the viral particles, you will also receive purified pAAV-TREtight-mTagBFP2-B19G plasmid DNA.

Helper virus for monosynaptic tracing with rabies virus; to be coinjected with pAAV-syn-FLEX-splitTVA-EGFP-tTA (Addgene# 100798). These AAV preparations are suitable purity for injection into animals.

Delivery

• Volume: 100 µL
• Titer: ≥ 1×10¹³ vg/mL
• Pricing: $375 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% Poloxamer 188
• Serotype: AAV1
• Purification: Iodixanol gradient ultracentrifugation
• Reporter Gene: BFP (in presence of Cre and 100798)

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

Terms and Licenses

• Academic/Nonprofit Terms:
  • Terms of Use for Viral Vectors
• Industry Terms:
  • Not Available to Industry

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

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.