Methodology article

A recombinase system facilitates cloning of expression cassettes in the ciliate Tetrahymena thermophila

Thomas Weide¹ , Ulrike Bockau^2,3 , Angelika Rave² , Lutz Herrmann^2,4 and Marcus WW Hartmann²

¹Universitaetskliniken Muenster (UKM), Abteilung für Molekulare Nephrologie, Domagkstr. 3a, D-48149 Muenster, Germany

²Cilian AG, Johann-Krane-Weg 42, D-48149 Muenster, Germany

³Institut für allgemeine Zoologie und Genetik, Universitaet Muenster, Schloßplatz 5, D-48149 Muenster, Germany

⁴Provendis GmbH, Eppinghofer Str. 50, 48468 Muelheim an der Ruhr, Germany

author email corresponding author email

BMC Microbiology 2007, 7:12doi:10.1186/1471-2180-7-12

Published:	1 March 2007

Abstract

Background

Tetrahymena thermophila is one of the best characterized unicellular eukaryotes and its genome is sequenced in its entirety. However, the AT-richness of the genome and an unusual codon usage cause problems in cloning and expression of the ciliate DNA. To overcome these technical hiatuses we developed a Cre-dependent recombinase system.

Results

We created novel donor and acceptor vectors that facilitate the transfer of expression cassettes from the donor into novel acceptor plasmid. Expression vectors were used that encode the 19 kDa C-terminus of the MSP1 protein of Plasmodium falciparum and a blasticidin S (bsdR) resistance gene, respectively. The functional expression of these genes was demonstrated by western blot analysis with MSP1 specific antibodies and by a blasticidin growing assay.

Conclusion

The Cre dependent recombinase system in combination with the modular structure of the donor vectors ease cloning and expression of foreign genes in the ciliate system, providing a powerful tool for protistology research in future.