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.

Ordering

Item	Catalog #	Description	Quantity	Price (USD)
Plasmid	100799	Standard format: Plasmid sent in bacteria as agar stab	1	$65	Add to Cart
AAV1	100799-AAV1	Virus (100 µL at titer ≥ 1×10¹³ vg/mL) and Plasmid. More Information	1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30	$380	Add to Cart

This material is available to academics and nonprofits only.

Backbone

• Vector backbone: pAAV
  (Search Vector Database)
• Backbone manufacturer: Stratagene
• Vector type: Mammalian Expression, AAV

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin
• 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

• Terms and Licenses:
  • UBMTA
  • Evrogen Limited Use Label License for FPs

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: $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

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

Resource Information

• Terms and Licenses:
  • Ancillary Agreement for Penn Vectors
  • Terms of Use for Viral Vectors

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.