Establishment of a Cre/loxP recombination system for N-terminal epitope tagging of genes in Tetrahymena

Clara Jana-Lui Busch,Alexander Vogt,Kazufumi Mochizuki

doi:10.1186/1471-2180-10-191

Clara Jana-Lui Busch, Alexander Vogt + Show 1 more

Open Access

https://doi.org/10.1186/1471-2180-10-191

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Abstract

BackgroundEpitope tagging is a powerful strategy to study the function of proteins. Although tools for C-terminal protein tagging in the ciliated protozoan Tetrahymena thermophila have been developed, N-terminal protein tagging in this organism is still technically demanding.ResultsIn this study, we have established a Cre/loxP recombination system in Tetrahymena and have applied this system for the N-terminal epitope tagging of Tetrahymena genes. Cre can be expressed in Tetrahymena and localizes to the macronucleus where it induces precise recombination at two loxP sequences in direct orientation in the Tetrahymena macronuclear chromosome. This Cre/loxP recombination can be used to remove a loxP-flanked drug-resistance marker from an N-terminal tagging construct after it is integrated into the macronucleus.ConclusionsThe system established in this study allows us to express an N-terminal epitope tagged gene from its own endogenous promoter in Tetrahymena.

Highlights

Epitope tagging is a powerful strategy to study the function of proteins
A protein of interest is tagged by introducing a tag at its C-terminus [3,4,5] because a drug-resistance marker, which must be introduced in proximity to the tag for the establishment of transgenic strains, rarely disturbs the gene promoter if it is inserted downstream of a target gene; the tagged protein can be expressed at its endogenous levels
Cre-recombinase localizes to the macronucleus in Tetrahymena To test if Cre-recombinase can be expressed in Tetrahymena, we designed an inducible expression system for Cre

Summary

Introduction

Epitope tagging is a powerful strategy to study the function of proteins. tools for C-terminal protein tagging in the ciliated protozoan Tetrahymena thermophila have been developed, N-terminal protein tagging in this organism is still technically demanding. Epitope tagging has been widely used for the analysis of protein localization, interaction, and function (reviewed in [1]) It is extremely useful in studying the proteins of the ciliated protozoan Tetrahymena thermophila because epitope tags can be introduced efficiently into endogenous chromosomal loci by homologous recombination in this organism [2]. There is a drawback to the N-terminal epitope tagging strategy in general: an insertion of a drug-resistance marker into the upstream region of a gene could disturb its promoter activity. This possibility is especially an issue in the Tetrahymena system because intergenic sequences are relatively short in this organism [6]. If homologous recombination occurs within the coding sequence, an epitope tag at the

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