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
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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.