Transformation of Tetrahymena thermophila by Electroporation
This protocol was adapted from “Culture and Manipulation of Tetrahymena,” Chapter 18, in Cells (eds. Spector et al.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1998. This three-volume set is now out of print; however, some of the microscopy methods were republished in Basic Methods in Microscopy, by David L. Spector and Robert D. Goldman.INTRODUCTION
This protocol describes a method for transformation of the Tetrahymena using electroporation. The vector is electroporated into cells after mating, where it is incorporated into the DNA of developing macronuclei. Because T. thermophila can be propagated indefinitely without conjugation, transformation of the macronucleus provides a way to obtain stable somatic transformants. DNA vectors transformed using this protocol include those containing drug-resistant versions of Tetrahymena genes (which replace endogenous genes via homologous recombination) as well as those containing rDNA replication origins. Cotransformation using these two vector types is also possible: Electroporated cells are selected for drug resistance conferred by the replicating vector for 10-15 generations, then screened for the gene replacement using another drug-resistant marker. Usually a few percent of the cells that are transformed by the replicating vector are cotransformed by the gene replacement vector.
