INTRODUCTIONThis protocol describes a highly efficient procedure for transforming the vegetative macronucleus of Paramecium tetraurelia by DNA microinjection. Any microinjected DNA will be replicated without the need for specific origins and can be stably maintained at a wide range of copy numbers throughout vegetative growth as minichromosomes that are formed in vivo by the addition of telomeric sequences to the ends of linear monomers and multimers. A variable fraction of the injected DNA also integrates into endogenous macronuclear chromosomes by nonhomologous recombination. Endogenous transcription signals are recognized, allowing appropriate regulation of gene expression. This technique is used for complementation cloning of genes altered in mutants and for expression of modified genes, e.g., green fluorescent protein (GFP) fusions. Microinjection of nonexpressible constructs at high copy numbers can also be used to specifically silence homologous endogenous genes by transgene-induced RNA interference. Note that transformed clones are somatic transformants, and therefore can be maintained only during the vegetative phase of the life cycle (<200 cell divisions), i.e., only as long as they do not enter sexual reproduction or senescence. Cells must be kept in continuous exponential growth by providing a constant supply of food; starving cells with a clonal age ≥20 divisions, since the last sexual event will inevitably trigger meiosis, resulting in the loss of the transformed macronucleus and its replacement by a new macronucleus that develops from the germline micronuclei.