Gene Transfer, Targeting
and Therapeutics Core

Salk Institute for Biological Studies - Gene Transfer, Targeting
and Therapeutics Core

Virtual Kits


Virtual kits are sets of helper viruses (for expressing TVA, rabies glycoprotein (G) and/or marker genes) and G-deleted rabies vectors, that when used together, allow for monosynaptic circuit tracing. Cre-dependent kits use helper viruses with cre-dependent expression such that monosynaptic inputs to cre-expressing cells can be labeled.

Each kit is designed with differences in user goals in mind. Typically these differences are related to tradeoffs between experimental goals that are not both optimized with the same helper viruses. For example, there is a tradeoff between the ability to unambiguously count the numbers of starter neurons (possible when a marker gene is expressed from the same helper virus as G) and efficiency of transsynaptic labeling (optimized when G is expressed alone from the helper virus). For a more complete consideration of these and related design issues please refer to Callaway and Luo, Journal of Neuroscience, 35: 8879-85. http://www.ncbi.nlm.nih.gov/pubmed/26085623

Kit

Helper Viruses

Rabies Virus

Starter cell marker expression

Input cell marker expression

Pros

Cons

Kit #1: “All-in-one helper virus”

AAV-EF1a-DIO-HTB

EnvA-G-Deleted Rabies-mCherry

Histone-tagged GFP, mCherry

mCherry

Ease of use, Starter cells marked

Low Efficiency

Kit #2: “Dual helper virus with starter label”

AAV-Ef1a-DIO-H2B-GFP-2A-oG, AAV-FLEX-TVA

EnvA-G-Deleted Rabies-mCherry

Histone-tagged GFP, mCherry

mCherry

Starter cells marked

Moderate efficiency

Kit #3: “Dual helper, no starter label”

AAV-CAG-FLEX-oG, AAV-FLEX-TVA-mCherry

EnvA-G-Deleted Rabies-eGFP

mCherry marks TVA but no marker for G-expressing starters

eGFP

Highest efficiency

Starter cells not marked

Definitions:

Local Leak = Small amounts of TVA “leak” expression are expected in cells that do not express Cre. This will result in direct infection with EnvA Rabies. These directly infected cells will express the marker gene from the rabies virus but no glycoprotein, making them indistinguishable from transsynaptically labeled input cells. Leak expression is problematic for analysis of local circuits but does not interfere with unambiguous detection of distant inputs.

Efficiency = This refers to the numbers of input cells labeled per starter cell. Use of oG rather than B19G, as well as high expression of G are expected to improve efficiency. Use of 2A elements to express additional genes from the same vector that expresses G will result in reduced expression of G and therefore less efficiency. Therefore greater efficiency is achieved using a helper virus that expresses G alone than one that also expresses a marker gene. But co-expression of a marker gene is necessary to identify G-expressing starter cells for quantitative input tracing.