High frequency vector-mediated transformation and gene replacement in Tetrahymena.
Recently, we developed a mass DNA-mediated transformation technique for the ciliated protozoan Tetrahymena thermophila that introduces transforming DNA by electroporation into conjugating cells. Other studies demonstrated that a neomycin resistance gene flanked by Tetrahymena H4-I gene regulatory sequences transformed Tetrahymena by homologous recombination within the H4-I locus when microinjected into the macronucleus. We describe the use of conjugant electrotransformation (CET) for gene replacement and for the development of new independently replicating vectors and a gene cassette that can be used as a selectable marker in gene knockout experiments. Using CET, the neomycin resistance gene flanked by H4-I sequences transformed Tetrahymena, resulting in the replacement of the H4-I gene or integrative recombination of the H4-I/neo/H4-I gene (but not vector sequences) in the 5' or 3' flanking region of the H4-I locus. Gene replacement was obtained with non-digested plasmid DNA but releasing the insert increased the frequency of replacement events about 6-fold. The efficiency of transformation by the H4-I/neo/H4-I selectable marker was unchanged when a single copy of the Tetrahymena rDNA replication origin was included on the transforming plasmid. However, the efficiency of transformation using CET increased greatly when a tandem repeat of the replication origin fragment was used. This high frequency of transformation enabled mapping of the region required for H4-I promoter function to within 333 bp upstream of the initiator ATG. Similarly approximately 300 bp of sequence downstream of the translation terminator TGA of the beta-tubulin 2 (BTU2) gene could substitute for the 3' region of the H4-I gene. This hybrid H4-I/neo/BTU2 gene did not transform Tetrahymena when subcloned on a plasmid lacking an origin of replication, but did transform at high frequency on a two origin plasmid. Thus, the H4-I/neo/BTU2 cassette is a selectable marker that can be used for gene knockout in Tetrahymena. As a first step toward constructing a vector suitable for cloning genes by complementation of mutations in Tetrahymena, we also demonstrated that the vector containing 2 origins and the H4-I/neo/BTU2 cassette can co-express a gene encoding a cycloheximide resistant ribosomal protein.
Gaertig
J
J
Department of Biology, University of Rochester, NY 14627.
Gu
L
L
Hai
B
B
Gorovsky
M A
MA
D000818:Animals; D016054:DNA, Protozoan / Q000235:genetics; D018274:Electroporation; D018390:Gene Targeting / Q000379:methods*; D017125:Genes, Protozoan / Q000235:genetics*; D005809:Genes, Regulator / Q000235:genetics; D005819:Genetic Markers; D005822:Genetic Vectors / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D018741:Replication Origin / Q000235:genetics; D013728:Terminator Regions, Genetic / Q000235:genetics; D016808:Tetrahymena thermophila / Q000235:genetics*; D014170:Transformation, Genetic; D014404:Tubulin / Q000235:genetics
D016054:DNA, Protozoan; D005819:Genetic Markers; D014404:Tubulin