Expanding the power of recombinase-based labeling to uncover cellular diversity

Abstract

Investigating the developmental, structural and functional complexity of mammalian tissues and organs depends on identifying and gaining experimental access to diverse cell populations. Here, we describe a set of recombinase-responsive fluorescent indicator alleles in mice that significantly extends our ability to uncover cellular diversity by exploiting the intrinsic genetic signatures that uniquely define cell types. Using a recombinase-based intersectional strategy, these new alleles uniquely permit non-invasive labeling of cells defined by the overlap of up to three distinct gene expression domains. In response to different combinations of Cre, Flp and Dre recombinases, they express eGFP and/or tdTomato to allow the visualization of full cellular morphology. Here, we demonstrate the value of these features through a proof-of-principle analysis of the central noradrenergic system. We label previously inaccessible subpopulations of noradrenergic neurons to reveal details of their three-dimensional architecture and axon projection profiles. These new indicator alleles will provide experimental access to cell populations at unprecedented resolution, facilitating analysis of their developmental origin and anatomical, molecular and physiological properties.

Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins

“…5a, b). Similarly, to determine whether the knock-in alleles express as desired, one Fgf8 P2A-FlpO founder and one Slc26a5 P2A-FlpO F1 were bred to a FlpO reporter line [30] and the offspring were analyzed for expression of tdTomato. As expected, the offspring of these two animals showed appropriate expression of the inserted sequence ( Fgf8 P2A-FlpO #4 drove expression in cochlear inner hair cells; Fig.…”

Recombinase-Dependent Mouse Lines for Chemogenetic Activation of Genetically Defined Cell Types

“…To demonstrate intersectional Flp/Cre control of RC::FL-hM3Dq , we targeted a population of noradrenergic neurons defined by a shared history of both En1 cre and Dbh Flpo expression (Robertson et al, 2013). This combination of recombinase drivers restricts hM3Dq-mCherry expression to a subpopulation of noradrenergic neurons (hereafter designated LC complex), which encompasses 99.8% of the compact locus coeruleus (LC) and a portion of the dorsal subcoeruleus and A7 noradrenergic nuclei (Robertson et al, 2013; Plummer et al, 2015). As expected, mCherry fluorescence was restricted to the soma and dendrites of the LC complex (Figure 3D, Figure S4).…”

“…We amplified FRT-flanked and rox-flanked His3-SV40 transcriptional stop cassettes, using as the PCR template a pBS302 plasmid (Sauer, 1993) modified to remove the internal MfeI site. The stop cassettes and FLEx switch containing eGFP were then cloned into the Gt(ROSA)26Sor targeting vector pRC-RFLTG (Plummer et al, 2015) after digestion with MluI and FseI. The digested fragment of pRC-RFLTG provided a CAG promoter (Niwa et al, 1991), woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) (Zufferey et al, 1999), bGH poly(A) cassette, the 5’ and 3’ homology to the Gt(ROSA)26Sor locus, and attB/attP-flanked Neo cassette.…”

A novel floor plate boundary defined by adjacentEn1andDbx1microdomains distinguishes midbrain dopamine and hypothalamic neurons

“…S2D,D′) (Bielle et al, 2005) and En1::Dre (Plummer et al, 2016) in combination with two separate reporter alleles RC::RLTG (Rosa-CAG-rox-FRT-loxPtdTomato-eGFP) and RC::NZG (Rosa-CAG-loxP-PGKNeo-FRTnlsLacZ-eGFP) ( Fig. 3A) (Plummer et al, 2015;Yamamoto et al, 2009). In such embryos, the intersectional reporter RC::RLTG produces expression of tdTomato after En1::Dre recombination, and eGFP after intersectional En1::Dre and Dbx1::Cre recombination events; RC::NZG expresses nls-lacZ after Dbx1:: Cre recombination events.…”