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.

A three part figure showing intersectional expression approaches. Part A shows three Venn-like diagrams depicting Cre AND Flp spheres with the overlapping area shaded, Cre NOT Flp spheres overlapping with the Cre area alone shaded, and finally Flp NOT Cre spheres with the Flp area alone shaded. Part B shows a three sphere overlap of Cre AND Flp AND VCre with only the overlapping area in the middle shaded. Part C shows six images of hippocampal slices, three at 500 micrometers and three at 50 micrometers. After injection with AAV-DJ-CON/FON-ChR2-eYFP, expression is only seen in the stratum oriens in PV-2a-Cre;SOM-IRES-Flp animals and not SOM-IRES-Flp or PV-2a-Cre animals. — 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.

A six part figure of the single (Parts A-C) and double (Parts D-F) intersectional constructs. Part A shows the three single intron intersectional constructs (each containing two orthogonal recombinase (Cre-dependent) recognition sequences flanking exon 1 and two orthogonal recombinase (Flp-dependent) recognition sequences flanking exon 2, with the four recognition sequences in the middle making up the intron): CON/FON (exon 1 (3’ to 5’), and exon 2 (3’ to 5’)), CON/FOFF (exon 1 (3’ to 5’), and exon 2), and COFF/FON (exon 1, and exon 2). Three example tool classes are also listed: fluorophores (ex. EYFP), GECIs (ex. GCaMP6m), and rabies glycoprotein (ex. OG). Part B shows each initial state (CON/FON, CON/FOFF, and COFF/FON,) with the addition of Cre and Flp, Cre, or Flp respectively leading to the directionally aligned exon 1 exon 2 active state. Addition of Cre leads to the COFF/FON inactivated state (exon 1 (3’ to 5’) exon 2) or the addition of Flp leads to the CON/FOFF inactivated state (exon 1 exon 2 (3’ to 5’). Part C shows the Cre-dependent and Flp-dependent parts of the construct as it moves from initial state to active state after recombinase activity (leaving single recognition sites flanking each exon), and from active state to mRNA after intron splicing (removing the intron made up of the two remaining recognition sites between the exons), and from mRNA to protein after translation. Part D shows the double intron intersection constructs (each containing two orthogonal recombinase (Cre-dependent) recognition sequences flanking exon 2 and two orthogonal recombinase (Flp-dependent) recognition sequences flanking all three exons, the two sets of recognition sequences between exons 3 and exons 2 make up intron 1 and the two sets of recognition sequences between exon 2 and exon 1 make up intron 2) CON/FON (exon 3 (3’ to 5’), exon 2, and exon 1 (3’ to 5’)), CON/FOFF (exon 3, exon 2 (3’ to 5’), and exon 1), and COFF/FON (exon 3 (3’ to 5’), exon 2 (3’ to 5’), and exon 1 (3’ to 5’)). Three example tool classes are also listed: Excitatory Opsins (ex. ChR2-EYFP), Inhibitory Opsins (ex. iC++-EYFP), and Rabies Glycoprotein (ex. TVA-mCherry). Part E shows each initial state (CON/FON, CON/FOFF, and COFF/FON,) with the addition of Cre and Flp, Cre, or Flp respectively leading to the directionally aligned exon 1 exon 2 exon 3 active state. With the addition of Cre we reach the COFF/FON inactivated state (exon 1 exon 2 (3’ to 5’) exon 3) or with the addition of Flp the CON/FOFF inactivated state (exon 3 (3’ to 5’)exon 2 (3’ to 5’) exon 1 (3’ to 5’)). Part F is similar to Part C but with the double introns. — 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.
The Fenno Lab maintains a public folder (Link opens in a new window) of manuscripts that have used these constructs experimentally.

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	In-Stock Viral Items
55636	pAAV-EF1a-Cre	None	Yes
55632	pAAV-Ef1a-mCherry-IRES-Cre	None	Yes
55637	pAAV-EF1a-Flpo	None	Yes
55634	pAAV-EF1a-mCherry-IRES-Flpo	None	Yes
55638	pAAV-EF1a-vCre	None	Yes
55635	pAAV-EF1a-sCre	None	No
55633	pAAV-EF1a-mCherry-IRES-Dre	None	No
183535	pAAV-CaMKIIa-Flpo	None	No

Single recombinase-dependent

Addgene ID	Plasmid	Logic	In-Stock Viral Items
55641	pAAV-Ef1a-fDIO EYFP	Flp	Yes
55640	pAAV-Ef1a-dDIO hChR2(H134R)-EYFP	Dre	No
55639	pAAV-Ef1a-fDIO hChR2(H134R)-EYFP	Flp	Yes
126080	pAAV-Ef1a-sCreDIO hChR2(H134R)-eYFP	Scre	No
126081	pAAV-Ef1a-vCreDIO hChR2(H134R)-eYFP	Vcre	No

Dual recombinase-dependent: Fluorophores

Addgene ID	Plasmid	Logic	Sites and Mutations	In-Stock Viral Items
55650	pAAV-hSyn Con/Fon EYFP	Cre AND Flp		Yes
231926	pAAV-Ef1a-Con/Fon-EYFP	Cre AND Flp		No
55651	pAAV-hSyn Con/Foff EYFP	Cre AND NOT Flp	F3/F5	No
137162	pAAV-Ef1a-Con/Foff 2.0-EYFP	Cre AND NOT Flp	FRT/F5	Yes
55652	pAAV-hSyn Coff/Fon EYFP	Flp AND NOT Cre		No
231927	pAAV-Ef1a-Coff/Fon-EYFP	Flp AND NOT Cre		Yes
137129	pAAV-Ef1a-Con/Fon-BFP	Cre AND Flp		Yes
137130	pAAV-Ef1a-Con/Foff 2.0-BFP	Cre AND NOT Flp	FRT/F5	Yes
137131	pAAV-Ef1a-Coff/Fon-BFP	Flp AND NOT Cre		Yes
137132	pAAV-Ef1a-Con/Fon-mCherry	Cre AND Flp		Yes
137133	pAAV-Ef1a-Con/Foff 2.0-mCherry	Cre AND NOT Flp	FRT/F5	Yes
137134	pAAV-Ef1a-Coff/Fon-mCherry	Flp AND NOT Cre		Yes
137135	pAAV-Ef1a-oScarlet	None		Yes
137136	pAAV-Ef1a-Con/Fon-oScarlet	Cre AND Flp		Yes
137137	pAAV-Ef1a-Con/Foff 2.0-oScarlet	Cre AND NOT Flp	FRT/F5	Yes
137138	pAAV-Ef1a-Coff/Fon-oScarlet	Flp AND NOT Cre		Yes

Dual recombinase-dependent: GECI

Addgene ID	Plasmid	Logic	Sites and Mutations	In-Stock Viral Items
137119	pAAV-EF1a-Con/Fon-GCaMP6M	Cre AND Flp		Yes
137120	pAAV-Ef1a-Con/Foff 2.0-GCaMP6M	Cre AND NOT Flp	FRT/F5	Yes
137121	pAAV-Ef1a-Coff/Fon-GCaMP6M	Flp AND NOT Cre		Yes
137122	pAAV-Ef1a-Con/Fon-GCaMP6F	Cre AND Flp		Yes
137123	pAAV-Ef1a-Con/Foff 2.0-GCaMP6F	Cre AND NOT Flp	FRT/F5	Yes
137124	pAAV-Ef1a-Coff/Fon-GCaMP6F	Flp AND NOT Cre		Yes
137125	pAAV-Ef1a-sRGECO	None		Yes
137126	pAAV-Ef1a-Con/Fon-sRGECO	Cre AND Flp		Yes
137127	pAAV-Ef1a-Con/Foff 2.0-sRGECO	Cre AND NOT Flp	FRT/F5	Yes
137128	pAAV-Ef1a-Coff/Fon-sRGECO	Flp AND NOT Cre		Yes

Dual recombinase-dependent: Excitatory Opsins

Addgene ID	Plasmid	Logic	Sites and Mutations	In-Stock Viral Items
55645	pAAV-hSyn Con/Fon hChR2(H134R)-EYFP	Cre AND Flp		Yes
55644	pAAV-nEF Con/Fon hChR2(H134R)-EYFP	Cre AND Flp		No
55647	pAAV-nEF Con/Foff hChR2(H134R)-EYFP	Cre AND NOT Flp	F3/F5	No
137163	pAAV-nEF-Con/Foff 2.0-ChR2-EYFP	Cre AND NOT Flp	FRT/F5	Yes
55646	pAAV-hSyn Con/Foff hChR2(H134R)-EYFP	Cre AND NOT Flp	F3/F5	No
55649	pAAV-hnEF Coff/Fon hChR2(H134R)-EYFP	Flp AND NOT Cre		No
55648	pAAV-hSyn Coff/Fon hChR2(H134R)-EYFP	Flp AND NOT Cre		No
137139	pAAV-nEF-Con/Fon-ChR2(ET/TC)-EYFP	Cre AND NOT Flp		Yes
137140	pAAV-nEF-Con/Foff 2.0-ChR2(ET/TC)-EYFP	Cre AND Flp	FRT/F5	Yes
137141	pAAV-nEF-Coff/Fon-ChR2(ET/TC)-EYFP	Flp AND NOT Cre		Yes
137142	pAAV-nEF-Con/Fon-ChR2-mCherry	Cre AND Flp		Yes
137143	pAAV-nEF-Con/Foff 2.0-ChR2-mCherry	Cre AND NOT Flp	FRT/F5	Yes
137144	pAAV-nEF-Coff/Fon-ChR2-mCherry	Flp AND NOT Cre		Yes
137145	pAAV-nEF-Con/Fon-bREACHes-EYFP	Cre AND Flp		No
137146	pAAV-nEF-Con/Foff 2.0-bREACHes-EYFP	Cre AND NOT Flp	FRT/F5	No
137147	pAAV-nEF-Coff/Fon-bREACHes-EYFP	Flp AND NOT Cre		No
137158	pAAV-nEF-ChRmine-mScarlet	None		Yes
137159	pAAV-nEF-Con/Fon-ChRmine-oScarlet	Cre AND Flp		Yes
137160	pAAV-nEF-Coff/Fon-ChRmine-oScarlet	Cre AND NOT Flp	FRT/F5	Yes
137161	pAAV-nEF-Con/Foff 2.0-ChRmine-oScarlet	Flp AND NOT Cre		Yes

Dual recombinase-dependent: Inhibitory Opsins

Addgene ID	Plasmid	Logic	Sites and Mutations	In-Stock Viral Items
137148	pAAV-nEF-Con/Fon-Arch3.3-p2a-EYFP	Cre AND Flp		Yes
137149	pAAV-nEF-Con/Foff 2.0-Arch3.3-EYFP	Cre AND NOT Flp	FRT/F5	Yes
137150	pAAV-nEF-Coff/Fon-Arch3.3-p2a-EYFP	Flp AND NOT Cre		Yes
137151	pAAV-nEF-NpHR3.3-EYFP	None	W179F	Yes
137152	pAAV-nEF-Con/Fon-NpHR3.3-EYFP	Cre AND Flp	W179F	Yes
137153	pAAV-nEF-Con/Foff 2.0-NpHR3.3-EYFP	Cre AND NOT Flp	FRT/F5; W179F	Yes
137154	pAAV-nEF-Coff/Fon-NpHR3.3-EYFP	Flp AND NOT Cre	W179F	Yes
137155	pAAV-nEF-Con/Fon-iC++-EYFP	Cre AND Flp		Yes
137156	pAAV-nEF-Con/Foff 2.0-iC++-EYFP	Cre AND NOT Flp	FRT/F5	Yes
137157	pAAV-nEF-Coff/Fon-iC++-EYFP	Flp AND NOT Cre		Yes

Dual recombinase-dependent: DREADDs

Addgene ID	Plasmid	Logic	In-Stock Viral Items
177669	pAAV-nEF-Coff/Fon DREADD Gi-mCherry	Flp AND NOT Cre	No
177672	pAAV-nEF-Con/Fon DREADD Gi-mCherry	Cre AND Flp	Yes
177673	pAAV-nEF-Con/Foff DREADD Gi-mCherry	Cre AND NOT Flp	Yes
183532	pAAV-nEF Con/Fon DREADD Gq-mCherry	Cre AND Flp	No
183533	pAAV-nEF Con/Foff DREADD Gq-mCherry	Cre AND NOT Flp	No
183534	pAAV-nEF Coff/Fon DREADD Gq-mCherry	Flp AND NOT Cre	No

Dual recombinase-dependent: Rabies-related

Addgene ID	Plasmid	Logic	Sites and Mutations	In-Stock Viral Items
131779	pAAV-nEF-Con/Fon TVA-mCherry	Cre AND Flp		No

Triple recombinase-dependent

Addgene ID	Plasmid	Logic	Sites and Mutations	In-Stock Viral Items
137164	pAAV-nEF-Con/Fon/Von-EYFP	Cre AND Flp AND Vcre		Yes
137165	pAAV-Ef1a-Con/Fon/Von-GCaMP6M	Cre AND Flp AND Vcre		No

References

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