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Intersectional Genetics: Cre-lox with a twist

Limitations to scientific progress often arise not from a lack of curiosity or imagination, but rather from the tools and technologies available. While some research questions can be sufficiently answered with a broad “hammer” approach, others require tools with greater precision and finesse. Intriguingly, a singular tool may serve as a “hammer” for some queries and as a more nuanced instrument for others.

The advent of Cre-lox recombinase technology in mouse models revolutionized the life sciences. It opened the door to elucidating gene function within a defined cell population in vivo. Cre-lox technology initially represented the pinnacle of sophistication; but as it drove scientific breakthroughs and advanced understanding, more refined questions arose, creating the need for an even more sophisticated tool.

Cre-lox technology works by defining a cell population by a single gene. However, as advancements have demonstrated, not all cells expressing a shared gene have identical roles within a biological system. This cellular heterogeneity has been challenging to investigate due to the absence of tools to selectively manipulate and examine specific cell subpopulations. Understanding the functional significance of heterogeneity within a system is essential for progressing biological knowledge, especially to identify targets for precision therapeutics and interventions.

One promising approach to overcome the challenge of selectivity is through intersectional genetics. This methodology facilitates spatial and temporal genome manipulation in a more precisely defined subset of cells. Building upon the innovative Cre-lox technology, intersectional genetics combines multiple recombinase systems (Cre, CreERT, Tet, Flp, Dre) in a single mouse. Each recombinase recognizes its own target sites for recombination (Cre-lox, CreERT-lox, Tet-tTA or Tet-rtTA, Flp-Frt, Dre-rox). This specificity allows for the expression of anatomical and functional reporter proteins, gene knockouts, and gene knock-ins in cell populations defined by the expression of a distinct combination of genes, rather than a single gene.

For example, a CckCre::Ai40D or a Slc32a1Cre::Ai40D mouse enables visualization and optogenetic stimulation of all Cck-expressing cells or all GABAergic-expressing neurons, respectively. Alternatively, with an intersectional approach, a CckCre::Slc32a1FlpO::Ai80D mouse enables selective visualization and optogenetic manipulation of the specific subpopulation of interest — GABAergic neurons coexpressing Cck. The additional control of temporal manipulation can be achieved by using CreERT or Tet as one of the recombinases.

Intersectional genetics is comprised of at minimum three elements: 1) a recombinase driver line of interest, 2) a different recombinase driver line of interest, and 3) a “double stop” reporter line that is recombined by the driver lines selected. Multiple recombinase systems can be incorporated into a single mouse model through traditional breeding and/or viral delivery. Although more time-consuming, the conventional breeding approach offers the advantage of consistent, reproducible expression over successive generations. On the other hand, viral delivery provides a more expedited means of introducing multiple recombinase systems in the mouse model. This approach may be particularly advantageous when time is a critical factor, but careful consideration must be given to factors such as variability in transduction efficiency.

The JAX repository has many driver and reporter models suitable for intersectional genetics. Below is a curated list of intersectional genetics reporter models. These models can be crossed with the appropriate driver mice, enabling selective labeling, dual-labeling, and/or manipulation of well-defined cellular subpopulations.

Have questions about utilizing intersectional genetics in your research? Contact our Technical Information Services group at micetech@jax.org. Need a refresher about Cre-lox technology or recombinase systems? Click here for an excellent overview.

Cre::Tet Dual Inducible Reporter Strains

JAX Strain # Common Name Promoter Effector Expression Strain Nomenclature Reference Availability
024108 Ai93 TIGRE :: TRE; CaMK2a GCaMP6f + tTA Forebrain calcium indicator STOCK Igs7tm93.1(tetO-GCaMP6f)Hze Tg(Camk2a-tTA)1Mmay/J Original

Cited

Cryo Recovery
031562 Ai162D TIGRE :: TRE2 + CAG GCaMP6s + tTA2s Cre- and Tet-dependent ; calcium indicator B6.Cg-Igs7tm162.1(tetO-GCaMP6s,CAG-tTA2)Hze/J Original

Cited

Live
030328 Ai148D TIGRE :: TRE2 + CAG GCaMP6f + tTA2 Cre- and Tet-dependent ; calcium indicator B6.Cg-Igs7tm148.1(tetO-GCaMP6f,CAG-tTA2)Hze/J Original

Cited

Live
024115 Ai94D TIGRE :: TRE ; CaMK2a GCaMP6s + tTA Forebrain calcium indicator before dox B6.Cg-Igs7tm94.1(tetO-GCaMP6s)Hze Tg(Camk2a-tTA)1Mmay/J Cited
(GCaMP6s)

Cited
(Camk2a)

Cryo Recovery
030217 Ai143D TIGRE :: TRE RCaMP1.07 Cre- and Tet-dependent ; calcium indicator [red] B6.Cg-Igs7tm143.1(tetO-RCaMP1.07)Hze/J Original

Cited

Cryo Recovery
030219 Ai139D TIGRE :: TRE2 + CAG EGFP + tdT + tTA2 Cre- and Tet-dependent ; differential xFP B6.Cg-Igs7tm139.1(tetO-EGFP,CAG-tdTomato,-tTA2)Hze/J Original

Cited

Cryo Recovery
030220 Ai140D TIGRE :: TRE2 + CAG EGFP + tTA2 Cre- and Tet-dependent ; xFP B6.Cg-Igs7tm140.1(tetO-EGFP,CAG-tTA2)Hze/J Original

Cited

Cryo Recovery
029679 Optopatch3 Ai155 TIGRE :: TRE CheRiff-eGFP :: QuasAr3-mCitrine Cre- & Tet-dependent ; all-optical electrophysiology B6;129S6-Igs7tm3(tetO-Optopatch3,CAG-tTA)Acoh/J Original

Cited

Cryo Recovery
031569 Ai169D TIGRE :: TRE2 + CAG ASAP2s + tTA2s Cre- and Tet-dependent ; voltage indicator B6.Cg-Igs7tm169.1(tetO-GFP*/TPTE2*,CAG-tTA2)Hze/J Original Cryo Recovery
034112 Ai195 TIGRE :: TRE2 + CAG jGCaMP7s + tTA2 Cre- and Tet- dependent ; EGFP calcium indicator B6;129S6-Igs7tm195(tetO-GCaMP7s,CAG-tTA2)Tasic/J Original Live
029633 Rosa26-CAGs-LSL-RIK knock-in CAG :: TRE mKate2 + rtTA3 Cre-inducible mKate2 fluorescence (far-red) ; then Dox-inducible (Tet-On) gene expression B6.Cg-Gt(ROSA)26Sortm2(CAG-rtTA3,-mKate2)Slowe/J Original

Cited

Live

Cre::Flp Dual Reporter Strains

JAX Strain # Common Name Promoter Effector Expression Strain Nomenclature Reference Availability
037382 Ai224 TIGRE :: CAG EGFP/tdTomato Cre-dependent EGFP expression; Flp-dependent tdTomato expression B6.Cg-Igs7tm224(CAG-EGFP,CAG-dTomato)Tasic/J Original Live
025109 Ai80D R26 :: CAG CatCh (ChR2*L132C) / EYFP Cre- and Flp-dependent ; opsin/xFP B6.Cg-Gt(ROSA)26Sortm80.1(CAG-COP4*L132C/EYFP)Hze/J Original

Cited

Cryo Recovery
024846 R26 LSL FSF ReaChR-mCitrine R26 :: CAG ReaChR / mCitrine Cre- and Flp-dependent ; opsin/xFP B6;129S-Gt(ROSA)26Sortm2.1Ksvo/J Original

Cited

Cryo Recovery
030206 RC :: FPSit R26 :: CAG Synaptophysin / EGFP + tdTomato Cre- and Flp-dependent ; xFP [synapse] B6;129S6-Gt(ROSA)26Sortm10(CAG-Syp/EGFP*,-tdTomato)Dym/J Original

Cited

Cryo Recovery
021875 Ai65D R26 :: CAG tdTomato Cre- and Flp-dependent ; xFP B6;129S-Gt(ROSA)26Sortm65.1(CAG-tdTomato)Hze/J Original

Cited

Live
026932 RC :: FLTG R26 :: CAG tdTomato; EGFP Flp-dependent tdT ; then Cre-dependent EGFP B6.Cg-Gt(ROSA)26Sortm1.3(CAG-tdTomato,-EGFP)Pjen/J Original

Cited

Live
029040 RC :: FPDi R26 :: CAG inhib. Gi-DREADD (hM4Di) :: mCherry Flp-inducible mCherry, then Cre- & CNO-inducible canonical Gi pathway (neuron silencing) B6;129S6-Gt(ROSA)26Sortm9(CAG-mCherry,-CHRM4*)Dym/J Original

Cited

Cryo Recovery
026942 RC :: FL-hM3Dq R26 :: CAG eGFP :: FLEx switch hM3Dq/mCherry Flp-inducible eGFP and RC::L-hM3Dq allele, then Cre-inducible hM3Dq-mCherry-2ACT88 fusion protein & CNO inducible hM3Dq activation (canonical Gq pathway, neuronal activity/neuronal firing) B6.Cg-Gt(ROSA)26Sortm3.2(CAG-EGFP,-CHRM3*/mCherry/Htr2a)Pjen/J Original

Cited

Live
036590 Cot2 / R26-EYFP R26 :: CAG Cot2 allele (Cre or Tomato on chromosome 2) + EGFP Flp-dependent & tamoxifen inducible expression of Cot2, then Cre-dependent expression of EYFP (introduces mosaicism to delineate cell-autonomous functions of any gene in different tissues) STOCK Gm10822Tn(pb-CAG-tdTomato,Cre)Cot2Zhu Gt(ROSA)26Sortm1(EYFP)Cos/J Original Live

Cre::Flp Dependent, Tet Inducible Reporter Strains

JAX Strain # Common Name Promoter Effector Expression Strain Nomenclature Reference Availability
034112 Ai195 TIGRE :: TRE2 + CAG jGCaMP7s (EGFP) + tTA2 Cre- and Flp- dependent jGCaMP7s EGFP and tTA2 expression, Dox inducible inhibition of tTA2 activity (reduce jGCaMP7s expression) B6;129S6-Igs7tm195(tetO-GCaMP7s,CAG-tTA2)Tasic/J Original Live
037379 Ai211 TIGRE :: tetO + CAG ChrR-tdT (light-gated cation channel ; neuronal activity) +tTA2 Cre- and Flp-dependent tTA2 and ChrR-tdT expression, Dox controllable expression of ChrR-tdT expression B6.Cg-Igs7tm211(tetO-ChrimsonR/tdTomato,CAG-tTA2)Tasic/J Original Cryo Recovery
037378 Ai210 TIGRE :: tetO + CAG jGCaMP7f (EGFP) + tTA2 Cre- and Flp- dependent jGCaMP7f EGFP and tTA2 expression, Dox inducible inhibition of tTA2 activity (reduce jGCaMP7f expression) B6.Cg-Igs7tm210(tetO-GCaMP7f,CAG-tTA2)Tasic/J Original Cryo Recovery