Mouse transgenic approaches in optogenetics

Abstract

A major challenge in neuroscience is to understand how universal behaviors, such as sensation, movement, cognition, and emotion, arise from the interactions of specific cells that are present within intricate neural networks in the brain. Dissection of such complex networks has typically relied on disturbing the activity of individual gene products, perturbing neuronal activities pharmacologically, or lesioning specific brain regions, to investigate the network's response in a behavioral output. Though informative for many kinds of studies, these approaches are not sufficiently fine-tuned for examining the functionality of specific cells or cell classes in a spatially or temporally restricted context. Recent advances in the field of optogenetics now enable researchers to monitor and manipulate the activity of genetically defined cell populations with the speed and precision uniquely afforded by light. Transgenic mice engineered to express optogenetic tools in a cell type-specific manner offer a powerful approach for examining the role of particular cells in discrete circuits in a defined and reproducible way. Not surprisingly then, recent years have seen substantial efforts directed toward generating transgenic mouse lines that express functionally relevant levels of optogenetic tools. In this chapter, we review the state of these efforts and consider aspects of the current technology that would benefit from additional improvement.

Figure 1

Single transgenic approaches to expressing optogenetic tools (using ChR2 as an example). (A) Conventional transgenic approach, in which an expression cassette contains a promoter and the transgene and is randomly integrated into the genome. GS Pr, gene-specific promoter. pA, polyA signal. (B) Gene trap approach, in which a promoterless cassette containing the transgene is randomly integrated into the genome, and the transgene expression is determined by a “trapped” nearby endogenous promoter. SA, splice acceptor. Black boxes indicate endogenous gene exons. (C) BAC transgenic approach, in which the transgene is inserted into the locus of the gene-of-interest contained within a BAC clone, and this BAC clone is randomly integrated into the genome. (D) Knock-in approach, in which the transgene is targeted to the endogenous locus of the gene-of-interest by homologous recombination. The targeting site can be either at the ATG start codon (upper panel), or at the STOP codon (lower panel).

Figure 2

The binary transgenic systems for expressing optogenetic tools (using ChR2 as an example). (A) The Cre/lox system, in which the driver line expresses Cre under the control of a gene-specific promoter, and the reporter line directs Cre-dependent expression of the transgene. Cre-mediated recombination between the two loxP sites deletes the STOP cassette and hence induces the transgene expression. UB Pr, ubiquitous promoter. (B) The Tet-inducible system, in which the driver line expresses tTA under the control of a gene-specific promoter, and the reporter line expresses the transgene under the TRE promoter. tTA binds to the TRE promoter (TRE Pr) to activate transcription of the transgene. Upon binding to tetracycline or doxycycline (Dox), tTA is released from the TRE promoter and transcription stops (bottom panel).

Figure 3

The intersectional approaches to expressing optogenetic tools (using ChR2 as an example) to higher specificity. (A) A simple intersectional approach, in which the driver line uses the gene-specific promoter 1 (GS Pr-1) and the reporter line uses the gene-specific promoter 2 (GS Pr-2). (B) A Cre/Flp dual recombinase intersectional approach, in which the Cre driver line uses gene-specific promoter 1, and the Flp driver line uses gene-specific promoter 2. The double reporter line is both Cre and Flp dependent. (C) A Cre/tTA intersectional approach, in which the tTA driver line uses gene-specific promoter 1, and the Cre driver line uses genespecific promoter 2. The double reporter uses the TRE promoter and is also Cre dependent. HZ - 23