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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.

intersectional expression approaches

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 20141 (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 lines2-9 and combinations of transgenic recombinase-expressing animal lines and retro-grade expressing viruses delivering additional recombinases10-14.

single and double intersectional constructs

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 with general principles for working with INTRSECT recombinases, viruses, and important controls to consider as part of experimental design.
  • How-to guide for the molecular design and testing of novel INTSRECT plasmids for groups interested in producing their own INTRSECT plasmids.
  • Online spreadsheet 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

Singe 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

Addgene ID Plasmid Logic Sites and Mutations
55645 pAAV-hSyn Con/Fon hChR2(H134R)-EYFP Cre AND Flp
55644 pAAV-nEF Con/Fon hChR2(H134R)-EYFP Cre AND Flp
55647 pAAV-nEF Con/Foff hChR2(H134R)-EYFP Cre AND NOT Flp F3/F5
137163 pAAV-nEF-Con/Foff 2.0-ChR2-EYFP Cre AND NOT Flp FRT/F5
55646 pAAV-hSyn Con/Foff hChR2(H134R)-EYFP Cre AND NOT Flp F3/F5
55649 pAAV-hnEF Coff/Fon hChR2(H134R)-EYFP Flp AND NOT Cre
55648 pAAV-hSyn Coff/Fon hChR2(H134R)-EYFP Flp AND NOT Cre
137139 pAAV-nEF-Con/Fon-ChR2(ET/TC)-EYFP Cre AND NOT Flp
137140 pAAV-nEF-Con/Foff 2.0-ChR2(ET/TC)-EYFP Cre AND Flp FRT/F5
137141 pAAV-nEF-Coff/Fon-ChR2(ET/TC)-EYFP Flp AND NOT Cre
137142 pAAV-nEF-Con/Fon-ChR2-mCherry Cre AND Flp
137143 pAAV-nEF-Con/Foff 2.0-ChR2-mCherry Cre AND NOT Flp FRT/F5
137144 pAAV-nEF-Coff/Fon-ChR2-mCherry Flp AND NOT Cre
137145 pAAV-nEF-Con/Fon-bREACHes-EYFP Cre AND Flp
137146 pAAV-nEF-Con/Foff 2.0-bREACHes-EYFP Cre AND NOT Flp FRT/F5
137147 pAAV-nEF-Coff/Fon-bREACHes-EYFP Flp AND NOT Cre
137158 pAAV-nEF-ChRmine-mScarlet None
137159 pAAV-nEF-Con/Fon-ChRmine-oScarlet Cre AND Flp
137160 pAAV-nEF-Coff/Fon-ChRmine-oScarlet Cre AND NOT Flp FRT/F5
137161 pAAV-nEF-Con/Foff 2.0-ChRmine-oScarlet Flp AND NOT Cre

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: 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/VCon-EYFP Cre AND Flp AND Vcre
137165 pAAV-Ef1a-Con/Fon/VCon-GCaMP6M Cre AND Flp AND Vcre

References

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  11. Lazaridis I, Tzortzi O, Weglage M, Märtin A, Xuan Y, Parent M, Johansson Y, Fuzik J, Fürth D, Fenno LE, Ramakrishnan C, Silberberg G, Deisseroth K, Carlén M, Meletis K. 2019. A hypothalamus-habenula circuit controls aversion. Mol. Psychiatry 24(9):1351-1368. PMID:30755721
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  13. Marcinkiewcz CA, Mazzone CM, D'Agostino G, Halladay LR, Hardaway JA, DiBerto JF, Navarro M, Burnham N, Cristiano C, Dorrier CE, Tipton GJ, Ramakrishnan C, Kozicz T, Deisseroth K, Thiele TE, McElligott ZA, Holmes A, Heisler LK, Kash TL. 2016. Serotonin engages an anxiety and fear-promoting circuit in the extended amygdala. Nature 537(7618):97-101. PMID:27556938
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