Establishment of a Cre/loxP recombination system for N-terminal epitope tagging of genes in Tetrahymena
Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Dr. Bohr-Gasse 3, A-1030 Vienna, Austria
BMC Microbiology 2010, 10:191 doi:10.1186/1471-2180-10-191Published: 13 July 2010
Epitope 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.
In 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.
The system established in this study allows us to express an N-terminal epitope tagged gene from its own endogenous promoter in Tetrahymena.