Abstract
ABSTRACTThe advent of modern single-cell biology has revealed the striking molecular diversity of cell populations once thought to be more homogeneous. This newly appreciated complexity has made intersectional genetic approaches essential to understanding and probing cellular heterogeneity at the functional level. Here, we build on previous knowledge to develop a simple adeno-associated virus (AAV)-based approach to define specific subpopulations of cells by Boolean exclusion logic (AND NOT). This expression by Boolean exclusion (ExBoX) system encodes for a gene of interest that is turned on by a particular recombinase (Cre or FlpO) and turned off by another. ExBoX allows for the specific transcription of a gene of interest in cells expressing only the activating recombinase, but not in cells expressing both. We show the ability of the ExBoX system to tightly regulate expression of fluorescent reporters in vitro and in vivo, and further demonstrate the adaptability of the system by achieving expression of a variety of virally delivered coding sequences in the mouse brain. This simple strategy will expand the molecular toolkit available for cell- and time-specific gene expression in a variety of systems.
Highlights
Advancements in our understanding of the mechanisms underlying biological processes have been greatly dependent on the development of new genetic tools
Summary statement Ubina et al describe the generation of a novel Associated Virus (AAV)-based intersectional approach to define and target specific subpopulations of cells in time and space via Expression by Boolean Exclusion (ExBoX)
To control expression and label subsets of cells with greater specificity using Cre and Flp recombinases, we set out to develop a simple AAV-based expression system governed by Boolean Exclusion logic (Expression by Boolean Exclusion or ExBoX)
Summary
Advancements in our understanding of the mechanisms underlying biological processes have been greatly dependent on the development of new genetic tools. In comparison to conventional transgenic strategies that select cellular targets based on expression of one particular promoter or driver, intersectional approaches provide tighter specificity by selecting targets based on the overlapping or sequential expression of multiple recombinases (Andersson-Rolf et al, 2017; Jensen & Dymecki, 2014; Robles-Oteiza et al, 2015). These intersectional strategies based on the combinatorial expression of two recombinase systems (Cre/lox and Flp/FRT) were first used in the mouse to perform fate mapping of previously elusive neural progenitors (Dymecki, Ray, & Kim 2010). With multiple recombinase systems driving the expression of reporter genes, targeting of subpopulations of cells can be achieved through intersectional (expressing all drivers) and subtractive (lacking expression of one or more drivers) strategies (Farago et al, 2006)
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