A transgenic mouse line for collecting ribosome-bound mRNA using the tetracycline transactivator system.

Laurel Drane,Leon G Reijmers,Mark R Mayford,Joshua A Ainsley

doi:10.3389/fnmol.2014.00082

Laurel Drane, Leon G Reijmers + Show 2 more

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https://doi.org/10.3389/fnmol.2014.00082

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Abstract

Acquiring the gene expression profiles of specific neuronal cell-types is important for understanding their molecular identities. Genome-wide gene expression profiles of genetically defined cell-types can be acquired by collecting and sequencing mRNA that is bound to epitope-tagged ribosomes (TRAP; translating ribosome affinity purification). Here, we introduce a transgenic mouse model that combines the TRAP technique with the tetracycline transactivator (tTA) system by expressing EGFP-tagged ribosomal protein L10a (EGFP-L10a) under control of the tetracycline response element (tetO-TRAP). This allows both spatial control of EGFP-L10a expression through cell-type specific tTA expression, as well as temporal regulation by inhibiting transgene expression through the administration of doxycycline. We show that crossing tetO-TRAP mice with transgenic mice expressing tTA under the Camk2a promoter (Camk2a-tTA) results in offspring with cell-type specific expression of EGFP-L10a in CA1 pyramidal neurons and medium spiny neurons in the striatum. Co-immunoprecipitation confirmed that EGFP-L10a integrates into a functional ribosomal complex. In addition, collection of ribosome-bound mRNA from the hippocampus yielded the expected enrichment of genes expressed in CA1 pyramidal neurons, as well as a depletion of genes expressed in other hippocampal cell-types. Finally, we show that crossing tetO-TRAP mice with transgenic Fos-tTA mice enables the expression of EGFP-L10a in CA1 pyramidal neurons that are activated during a fear conditioning trial. The tetO-TRAP mouse can be combined with other tTA mouse lines to enable gene expression profiling of a variety of different cell-types.

Highlights

Acquiring the gene expression profiles of individual cell-types is imperative for understanding their molecular identities
GENERATION OF A MOUSE LINE THAT EXPRESSES EGFP-L10a UNDER CONTROL OF THE tetO PROMOTER We generated a transgenic mouse expressing the ribosomal protein L10a tagged with EGFP under the control of the tetracycline operon
In order to expand the arsenal of translating ribosome affinity purification (TRAP)-based tools for isolating ribosome-bound mRNA from defined cell types, we have generated a novel mouse line in which EGFP-L10a expression is under the control of the tetO promoter

Summary

Introduction

Acquiring the gene expression profiles of individual cell-types is imperative for understanding their molecular identities. Traditionally cell types are defined by their morphology and functional properties, recent studies have shown that many subclasses of neurons can be distinguished through genome-wide gene expression profiling (Okaty et al, 2011). These studies used various techniques to collect mRNA from defined groups of neurons, including laser capture microdissection (LCM) and fluorescence-activated cell sorting (FACS). Use of manual cell dissociation techniques can eliminate the off-target contamination seen with LCM This technique can be very time consuming as large numbers of cells must be isolated in order to collect sufficient mRNA quantities. The harsh conditions of the dissociation and sorting process causes neurons to lose their processes and increases the risk of introducing gene expression artifacts (Okaty et al, 2011)

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