Tetrahymena thermophila has emerged as an excellent protist model for studies on cilia that are based on reverse genetic approaches. In Tetrahymena, genes can be routinely disrupted by the DNA homologous recombination. We present established protocols for the manipulation of genes in either the germline micronucleus or the somatic macronucleus. A detailed protocol is provided for the construction of heterokaryon strains that carry a gene disruption only in the micronucleus. Heterokaryon strain can be propagated like wild-type cells, and ciliary phenotypes can be expressed on demand by mating. We describe methods that can be used for disruption of multiple genes. We include protocols for the generation and maintenance of Tetrahymena cells that either lack cilia or have paralyzed cilia.
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