INTRSECT Plasmid Collection
The development of powerful molecular reagents for biology have propelled our understanding of neural circuit functionality. Precisely expressing these tools in well-defined cellular sub-populations has generally been limited to single-component cellular definitions (e.g. neurons defined by a single gene or projection). Intersectional expression approaches combine multiplexed recombinases, including Cre, Flp, and VCre to enable viral expression of molecular payloads in target populations defined by multiple parameters.
Figure 1: Examples of intersectional cell population definitions using recombinases. A) Cre and Flp are multiplexed to enable intersectional viral targeting of populations expressing only Cre AND Flp, Cre NOT Flp, or Flp NOT Cre. B) This strategy has been expanded to incorporate VCre in order to enable three-feature cell targeting. C) Example of viral targeting of cells expressing Cre AND Flp, here expressed through a double transgenic animal strategy (PV-2a-Cre; SOM-IRES-Flp). Image from Fenno et al., 2014.
INTRSECT
INTRSECT (intronic recombinase sites enabling combinatorial targeting) is a synthetic molecular targeting strategy that allows adeno-associated virus (AAV)-borne payloads to be expressed in cells based on a doubly-specified combination of genetic and/or anatomical-defined parameters, by placing two orthogonal recombinase (Cre and Flp) recognition sequences within synthetic introns. INTRSECT was first shown as a proof-of-concept targeting approach in 2014 1 (using EYFP and ChR2-EYFP as payloads). This approach has been broadly applied using multiple recombinase-expression strategies to define cellular sub-populations of interest, including dual-transgenic recombinase-expressing mouse lines 2–9 and combinations of transgenic recombinase-expressing animal lines and retro-grade expressing viruses delivering additional recombinases 10–14.
Figure 2: INTRSECT works by inserting short, intronic sequences into the open reading frame (ORF) of a molecular tool and adding recombinase recognition sequences (e.g. Lox sites, FRT sites) inside of the introns (A,D). The addition of these recombinase recognition sequences enables directional control of the portion of the ORF that is sandwiched between the sites; the starting direction of these ORF fragments (which have become exons) determines the logical expression requirements of Cre and Flp (B,E). When the correct combinations of recombinases are present, the introns are excised during mRNA processing, producing a functional molecular tool (C,F).
Implementation
The following resources may be of interest for groups interesting in implementing intersectional experimental design.
- The Deisseroth Lab maintains a Standard Operating Procedure (Link opens in a new window) with general principles for working with INTRSECT recombinases, viruses, and important controls to consider as part of experimental design.
- How-to guide (Link opens in a new window) for the molecular design and testing of novel INTSRECT plasmids for groups interested in producing their own INTRSECT plasmids.
- Online spreadsheet (Link opens in a new window) of Flp-recombinase-expressing transgenic mouse and rat lines.
Plasmids
In addition to EYFP and ChR2-EYFP, a large number of additional, validated molecular payloads in the INTRSECT configuration have been developed, including additional fluorophores, excitatory and inhibitory opsins, genetically-encoded calcium indicators, and rabies targeting genes.
Recombinases
| Addgene ID | Plasmid | Logic | Sites and Mutations |
|---|---|---|---|
| 55636 | pAAV-EF1a-Cre | None | |
| 55632 | pAAV-Ef1a-mCherry-IRES-Cre | None | |
| 55637 | pAAV-EF1a-Flpo | None | |
| 55634 | pAAV-EF1a-mCherry-IRES-Flpo | None | |
| 55638 | pAAV-EF1a-vCre | None | |
| 55635 | pAAV-EF1a-sCre | None | |
| 55633 | pAAV-EF1a-mCherry-IRES-Dre | None | |
| 183535 | pAAV-CaMKIIa-Flpo | None |
Single recombinase-dependent
| Addgene ID | Plasmid | Logic | Sites and Mutations |
|---|---|---|---|
| 55641 | pAAV-Ef1a-fDIO EYFP | Flp | |
| 55640 | pAAV-Ef1a-dDIO hChR2(H134R)-EYFP | Dre | |
| 55639 | pAAV-Ef1a-fDIO hChR2(H134R)-EYFP | Flp | |
| 126080 | pAAV-Ef1a-sCreDIO hChR2(H134R)-eYFP | Scre | |
| 126081 | pAAV-Ef1a-vCreDIO hChR2(H134R)-eYFP | Vcre |
Dual recombinase-dependent: Fluorophores
| Addgene ID | Plasmid | Logic | Sites and Mutations |
|---|---|---|---|
| 55650 | pAAV-hSyn Con/Fon EYFP | Cre AND Flp | |
| 55651 | pAAV-hSyn Con/Foff EYFP | Cre AND NOT Flp | F3/F5 |
| 137162 | pAAV-Ef1a-Con/Foff 2.0-EYFP | Cre AND NOT Flp | FRT/F5 |
| 55652 | pAAV-hSyn Coff/Fon EYFP | Flp AND NOT Cre | |
| 137129 | pAAV-Ef1a-Con/Fon-BFP | Cre AND Flp | |
| 137130 | pAAV-Ef1a-Con/Foff 2.0-BFP | Cre AND NOT Flp | FRT/F5 |
| 137131 | pAAV-Ef1a-Coff/Fon-BFP | Flp AND NOT Cre | |
| 137132 | pAAV-Ef1a-Con/Fon-mCherry | Cre AND Flp | |
| 137133 | pAAV-Ef1a-Con/Foff 2.0-mCherry | Cre AND NOT Flp | FRT/F5 |
| 137134 | pAAV-Ef1a-Coff/Fon-mCherry | Flp AND NOT Cre | |
| 137135 | pAAV-Ef1a-oScarlet | None | |
| 137136 | pAAV-Ef1a-Con/Fon-oScarlet | Cre AND Flp | |
| 137137 | pAAV-Ef1a-Con/Foff 2.0-oScarlet | Cre AND NOT Flp | FRT/F5 |
| 137138 | pAAV-Ef1a-Coff/Fon-oScarlet | Flp AND NOT Cre |
Dual recombinase-dependent: GECI
| Addgene ID | Plasmid | Logic | Sites and Mutations |
|---|---|---|---|
| 137119 | pAAV-EF1a-Con/Fon-GCaMP6M | Cre AND Flp | |
| 137120 | pAAV-Ef1a-Con/Foff 2.0-GCaMP6M | Cre AND NOT Flp | FRT/F5 |
| 137121 | pAAV-Ef1a-Coff/Fon-GCaMP6M | Flp AND NOT Cre | |
| 137122 | pAAV-Ef1a-Con/Fon-GCaMP6F | Cre AND Flp | |
| 137123 | pAAV-Ef1a-Con/Foff 2.0-GCaMP6F | Cre AND NOT Flp | FRT/F5 |
| 137124 | pAAV-Ef1a-Coff/Fon-GCaMP6F | Flp AND NOT Cre | |
| 137125 | pAAV-Ef1a-sRGECO | None | |
| 137126 | pAAV-Ef1a-Con/Fon-sRGECO | Cre AND Flp | |
| 137127 | pAAV-Ef1a-Con/Foff 2.0-sRGECO | Cre AND NOT Flp | FRT/F5 |
| 137128 | pAAV-Ef1a-Coff/Fon-sRGECO | Flp AND NOT Cre |
Dual recombinase-dependent: Excitatory Opsins
Dual recombinase-dependent: Inhibitory Opsins
| Addgene ID | Plasmid | Logic | Sites and Mutations |
|---|---|---|---|
| 137148 | pAAV-nEF-Con/Fon-Arch3.3-p2a-EYFP | Cre AND Flp | |
| 137149 | pAAV-nEF-Con/Foff 2.0-Arch3.3-EYFP | Cre AND NOT Flp | FRT/F5 |
| 137150 | pAAV-nEF-Coff/Fon-Arch3.3-p2a-EYFP | Flp AND NOT Cre | |
| 137151 | pAAV-nEF-NpHR3.3-EYFP | None | W179F |
| 137152 | pAAV-nEF-Con/Fon-NpHR3.3-EYFP | Cre AND Flp | W179F |
| 137153 | pAAV-nEF-Con/Foff 2.0-NpHR3.3-EYFP | Cre AND NOT Flp | FRT/F5; W179F |
| 137154 | pAAV-nEF-Coff/Fon-NpHR3.3-EYFP | Flp AND NOT Cre | W179F |
| 137155 | pAAV-nEF-Con/Fon-iC++-EYFP | Cre AND Flp | |
| 137156 | pAAV-nEF-Con/Foff 2.0-iC++-EYFP | Cre AND NOT Flp | FRT/F5 |
| 137157 | pAAV-nEF-Coff/Fon-iC++-EYFP | Flp AND NOT Cre |
Dual recombinase-dependent: DREADDs
| Addgene ID | Plasmid | Logic | Sites and Mutations |
|---|---|---|---|
| 177669 | pAAV-nEF-Coff/Fon DREADD Gi-mCherry | Flp AND NOT Cre | |
| 177672 | pAAV-nEF-Con/Fon DREADD Gi-mCherry | Cre AND Flp | |
| 177673 | pAAV-nEF-Con/Foff DREADD Gi-mCherry | Cre AND NOT Flp | |
| 183532 | pAAV-nEF Con/Fon DREADD Gq-mCherry | Cre AND Flp | |
| 183533 | pAAV-nEF Con/Foff DREADD Gq-mCherry | Cre AND NOT Flp | |
| 183534 | pAAV-nEF Coff/Fon DREADD Gq-mCherry | Flp AND NOT Cre |
Dual recombinase-dependent: Rabies-related
| Addgene ID | Plasmid | Logic | Sites and Mutations |
|---|---|---|---|
| 131779 | pAAV-nEF-Con/Fon TVA-mCherry | Cre AND Flp | |
| pAAV-nEF-Con/Fon OG | Cre AND Flp |
Triple recombinase-dependent
| Addgene ID | Plasmid | Logic | Sites and Mutations |
|---|---|---|---|
| 137164 | pAAV-nEF-Con/Fon/Von-EYFP | Cre AND Flp AND Vcre | |
| 137165 | pAAV-Ef1a-Con/Fon/Von-GCaMP6M | Cre AND Flp AND Vcre |
References
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