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

Proc Natl Acad Sci U S A. 1997 Feb 18;94(4):1310-5. doi: 10.1073/pnas.94.4.1310.


The haploid Tetrahymena thermophila genome contains a single alpha-tubulin (ATU) gene. Using biolistic transformation, we disrupted one of the two copies of the ATU 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 the ATU 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 alpha-tubulin genes. However, when these conjugants were transformed with a functional marked ATU gene, viable transformants were obtained that contained the transforming ATU gene at the homologous locus in the new macronucleus. The exconjugant progeny could be rescued at a high efficiency (900 transformants per microg of DNA) with a wild-type ATU gene. Unlike previous macronuclear transformation protocols, this strategy should allow introduction of highly disadvantageous (but viable) mutations into Tetrahymena, 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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Cell Fusion
  • Cell Nucleus / genetics*
  • Crosses, Genetic
  • Gene Dosage
  • Gene Transfer Techniques
  • Genes, Lethal
  • Genes, Protozoan*
  • Genetic Vectors
  • Genome, Protozoan
  • Heterozygote
  • Homozygote
  • Micronucleus, Germline / genetics*
  • Mutation
  • Ploidies
  • Tetrahymena thermophila / genetics*
  • Transformation, Genetic
  • Tubulin / genetics*


  • Tubulin