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


CONCLUSIONS
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.
Weide
Thomas
T
Universitaetskliniken Muenster (UKM), Abteilung für Molekulare Nephrologie, Domagkstr, 3a, D-48149 Muenster, Germany. weidet@uni-muenster.de
Bockau
Ulrike
U
Rave
Angelika
A
Herrmann
Lutz
L
Hartmann
Marcus W W
MW
D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics / Q000378:metabolism; D004351:Drug Resistance / Q000235:genetics; D005069:Evaluation Studies as Topic; D005822:Genetic Vectors; D019426:Integrases; D020066:Merozoite Surface Protein 1 / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D010963:Plasmodium falciparum / Q000235:genetics; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D011760:Pyrrolidinones / Q000494:pharmacology; D011995:Recombination, Genetic; D016808:Tetrahymena thermophila / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*
D018076:DNA, Complementary; D016054:DNA, Protozoan; D020066:Merozoite Surface Protein 1; D015800:Protozoan Proteins; D011760:Pyrrolidinones; C420726:blasticidin A; C045073:Cre recombinase; D019426:Integrases