Germ-line knockout heterokaryons of an essential α-tubulin gene enable high-frequency gene replacement and a test of gene transfer from somatic to germ-line nuclei in Tetrahymena thermophila

The haploidTetrahymena thermophila genome contains a single α-tubulin (ATU ) gene. Using biolistic transformation, we disrupted one of the two copies of theATU gene in the diploid germ-line micronucleus. The heterozygous germ-line transformants were made homozygous in the micronucleus by mating to a star strain containing a defective micronucleus. This mating, known as round 1 genomic exclusion, resulted in two heterokaryon clones of different mating types which have both copies of theATU gene knocked out in the micronucleus but only wild-type genes in the polycopy somatic macronucleus. When these heterokaryons were mated, the exconjugant progeny cells did not grow because the new somatic macronuclei do not have any α-tubulin genes. However, when these conjugants were transformed with a functional markedATU gene, viable transformants were obtained that contained the transformingATU gene at the homologous locus in the new macronucleus. The exconjugant progeny could be rescued at a high efficiency (900 transformants per μg of DNA) with a wild-typeATU gene. Unlike previous macronuclear transformation protocols, this strategy should allow introduction of highly disadvantageous (but viable) mutations intoTetrahymena , providing a powerful tool for molecular and functional studies of essential genes. These knockout heterokaryons were used to demonstrate that gene transfer from somatic macronuclei to germ-line micronuclei occurs rarely if at all.