High frequency vector-mediated transformation and gene replacement in tetrahymena

Abstract

Recently, we developed a mass DNA-mediated transformation technique for the ciliated protozoan Tetrahymena thermophlla that Introduces transforming DNA by electroporatlon Into conjugating cells (1). Other studies demonstrated that a neomycln resistance gene flanked by Tetrahymena H4-I gene regulatory sequences transformed Tetrahymena by homologous recombination within the H4-I locus when mlcroinjected Into the macronucleus (2). We describe the use of conjugant electrotransformatlon (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 neomycln resistance gene flanked by H4-I sequences transformed Tetrahymena, resulting In the replacement of the H4-I gene or Integrative recombination of the H4-l/neo/H4-l 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-l7sol;neo/H4-l 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 promotor function to within 333 bp upstream of the Initiator ATG. Similarly ∼ 300 bp of sequence downstream of the translation terminator TGA of the /3-tubulin 2 (BTU2) gene could substitute for the 3' region of the H4-I gene. This hybrid H4-l/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-l/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-l/neo/BTU2 cassette can co-express a gene encoding a cyclohexlmlde resistant ribosomal protein. © 1994 Oxford University Press.