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Conditional Gene Expression and Targeting in Neuroscience Research

Alexei Morozov¹

¹Unit on Behavioral Genetics, Laboratory of Molecular Pathophysiology, National Institute of Mental Health, Bethesda, Maryland

Publication Name:

Current Protocols in Neuroscience

Unit Number:

Unit 4.31

DOI:

10.1002/0471142301.ns0431s44

Online Posting Date:

July, 2008

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Abstract

Recently developed techniques for spatially and temporally controlled genetic manipulations based on regulated homologous recombination and/or transcription are extensively used in brain research. In addition to being important for testing the role of specific proteins in the central nervous system, these techniques allow analysis of brain functions at the neuronal circuit level. This overview discusses principles of conditional inactivation and expression of genes, and their specific applications to studies of the mammalian brain. Curr. Protoc. Neurosci. 44:4.31.1-4.31.10. © 2008 by John Wiley & Sons, Inc.

Keywords: brain; conditional mutagenesis; Cre-loxP; tetracycline transactivator

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Introduction
Literature Cited
Figures

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Figures

Figure 4.31.1

Principle of conditional genetic manipulation. A gene of interest is modified to include a target sequence (solid triangle) recognized by a product of a gene regulator whose activity can be pharmacologically modulated. Expression of the gene regulator is driven by a region-specific promoter.

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Figure 4.31.2

Cre-mediated recombination. Cre recombinase deletes DNA sequences flanked by the loxP sites in the same orientation and inverts sequences flanked by the loxP sites in the opposite orientation.

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Figure 4.31.3

Cre-recombinase induced by steroid hormone analogs. Estrogen receptor-Cre chimeric protein (ER-Cre) remains in the cytoplasm until tamoxifen disrupts its binding to HSP70, allowing its translocation into the nucleus.

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Figure 4.31.4

Three-loxP targeting strategy. Targeting construct contains floxed critical exon and a cassette with positive (NEO) and negative (TK) selectable markers. Following transient Cre expression, the ES clones containing floxed critical exons (partial deletion) and those with a deletion of all floxed sequences (complete deletion) survive gancyclovir selection.

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Figure 4.31.5

Combination of Cre and Flp recombinases. Targeting construct with floxed critical exon contains a selectable marker (NEO) flanked by Frt sites, which allow the removal of the marker by Flp recombinase transiently expressed in ES cells or by using a deleter mouse.

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Figure 4.31.6

Conditional gene inactivation combined with expression of a reporter gene. The first exon of a gene is replaced with a floxed cDNA of interest followed by a floxed selectable marker (NEO) and a reporter (tau-lacZ). The marker is removed in the ES cells. Expression of the tau-lacZ reporter occurs only in those cells where cDNA is excised by Cre.

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Figure 4.31.7

tTA-regulated expression. (A) tTA binds to tet-O operator and drives transcription. The binding is blocked by doxycycline (DOX), which prevents tTA-dependent transcription. (B) Bidirectional tet-O promoter allows simultaneous expression of two genes, one of which is a reporter.

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Figure 4.31.8

Examples of tTA and Cre combinations. (A) Combinatorial restriction of gene expression by a combination of two gene regulators driven by promoters with overlapping patterns of expression. (B) Cre activates expression of tTA, which drives transcription of gene X. The expression pattern of gene X is restricted to the area where both promoter 1, driving expression of tTA, and promoter 2, driving expression of Cre, are active. (C) Cre expression is controlled by tTA driven by a region-specific promoter.

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Figure 4.31.9

Brainbow strategies for labeling of neighboring cells with different colors. (A) Three sets of incompatible lox sites (loxN, lox2272, and loxP) create three recombination possibilities (1, 2, or 3), switching OFP expression to RFP, YFP, or CFP. (B) Cre triggers inversion of a DNA segment flanked by loxP sites in opposite orientation, switching expression between RFP and M-CFP. Reproduced with permission from Livet et al. (2007).

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Figure 4.31.10

Tagging of neurons activated during a defined time window. tet-O-Tagged mice are raised on food containing DOX (left shaded block). During this time neuronal activation (lightning bolt) that leads to expression of tTA through c-fos promoter activation will not trigger tagging (neuron A), because DOX blocks activation of the tet-O promoter. The time window for tagging is opened by switching mice to food without DOX (middle white block). Neuronal activation will now activate the transcriptional feedback loop and start expression of tau-LacZ (neuron B). The time window is closed by putting mice back on DOX food (right dark shaded block) to block further feedback loop activation (neuron C). However, activation through the DOX-insensitive tTA (tTA^*) will continue to express tau-LacZ. Reproduced with permission from Reijmers et al. (2007).

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Literature Cited

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