by Martin Gorovsky
Efficient DNA-mediated transformation of Tetrahymena cells has been accomplished by three methods: microinjection , electrotransformation  and biolistic bombardment . Both the germline nucleus (micronucleus) or the somatic nucleus (macronucleus) can be transformed. (Click here for a description of the two types of nuclei.) Fig. 1 below shows the optimum life cycle stages for various types of transformation. By the appropriate choice of vectors and conditions, the incoming plasmid can be autonomously replicated in the MAC for many fissions, or the plasmid DNA can be inserted at the appropriate chromosome location by exact insert-mediated homologous recombination. The latter phenomenon allows gene replacements and knockouts, either in the MAC [ref.4; Fig. 2 below] or the MIC (ref.5; Fig. 3 below). Replacements and knockout strains are extremely valuable experimental tools, including the testing the indispensability of cloned genes and isolation of mutants after random mutagenesis of cloned genes. (Click here for a general Introduction to Tetrahymena Genetics.)
1. Tondravi,MM; Yao,M-C (1986): Transformation of Tetrahymena thermophila by microinjection of ribosomal RNA genes. Proc. Natl. Acad. Sci. USA 83, 4369-4373.
2. Gaertig,J; Gorovsky,MA (1992): Efficient mass transformation of Tetrahymena thermophila by electroporation of conjugants. Proc. Natl. Acad. Sci. USA 89, 9196-9200.
3. Cassidy-Hanley,D; Bowen,J; Lee,JH; Cole,E; VerPlank,LA; Gaertig,J; Gorovsky,MA; Bruns,PJ (1997): Germline and somatic transformation of mating Tetrahymena thermophila by particle bombardment. Genetics 146, 135-147.
4. Gaertig,J; Thatcher,TH; Gu,L; Gorovsky,MA (1994): Electroporation-mediated replacement of a positively and negatively selectable beta-tubulin gene in Tetrahymena thermophila. Proc. Natl. Acad. Sci. U. S. A. 91, 4549-4553.
5. Hai,B; Gorovsky,MA (1997): 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. 94, 1310-1315.
Hours indicate time after mixing of starved cells to initiate conjugation. BGg, germline transformation with the biolistic gun; BGc, conjugant transformation of macronuclei with the biolistic gun; CET, conjugant electrotransformation of developing macronuclei; BGv, vegetative macronuclei transformation with the biolistic gun; INJc, microinjection of developing macronuclei; INJv, microinjection of vegetative macronuclei.
Selection for complete gene replacement by phenotypic assortment enables the distinction between essential and non-essential genes. The initial transformant contains one transformed gene which has been disrupted with an expression cassette containing a drug-selectable marker. Its clonal progeny are serially transferred to higher drug concentrations thus killing cells with fewer copies of the selectable markers. For a non-essential gene (top diagram), complete replacement is possible. For an essential gene (bottom diagram), only incompletely assorted cells whose macronuclear copies of the gene have been partially replaced can be obtained. All arrows indicate multiple generations.
Creation and testing of knockout heterokaryons homozygous for disrupted ATU (alpha tubulin) genes in the micronucleus (deltaATU) and containing only wild type ATU genes in the macronucleus . Chx is a dominant gene conferring resistance to cycloheximide (cy-r) while the wild type Chx+ is sensitive (cy-s). Mpr is a dominant gene conferring resistance to 6-methylpurine (mp-r) to which the wild type Mpr+ gene is sensitive (mp-s). Roman numerals indicate mating types; mating types not determined are indicated by ?. Crosses (a), (b) and (d) are normal crosses, while cross (c) is a genomic exclusion cross (see Fig. 4 below for explanation of genomic exclusion). Note that in cross (d) only a mutant deltaATU/deltaATU Round I product of cross (c) is shown. An approximately equal number of non-mutant ATU/ATU Round I cells are obtained that do not yield any pm-r progeny. The genotype of the Mpr locus in Round I exconjugants was not determined. Values obtained for test crosses (b) and (d) are from a single subline.
* ("star") strains, have defective micronuclei; they can form conjugal pairs, but cannot donate genetic material [Allen,SL (1967) Genetics 55, 797-822]. When mated to a "star" strain, a normal strain ("left" conjugant, round 1) donates a gametic nucleus, but receives nothing in return. The single haploid nucleus in each conjugant then diploidizes and most cells separate without forming a new macronucleus. These cells retain their old (macronuclear) phenotype and are mature (can be immediately re-mated) but can have new micronuclear genotypes, depending on the genetic make-up of the normal parent and which meiotic product was (randomly) selected to form gametic nuclei. This process, referred to as Round 1 of genomic exclusion, greatly facilitates complementation testing and mutant rescue by cytoplasmic exchange during conjugation. Round 1 genomic exclusion or phenotypic assortment of heterozygotes each allow production of heterokaryons, cells with different macro- and micronuclear genotypes. Heterokaryons homozygous or heterozygous for a drug resistance gene in the transcriptionally inert micronucleus but having only drug-sensitive alleles in the macronucleus are drug sensitive. When these cells conjugated, the drug sensitive macronucleus is discarded and the drug resistance allele is expressed in the new macronucleus, allowing simple drug selection for successful mating. Round 1 genomic exclusion also allows creation of strains homozygous for deleterious or even lethal genes in their micronuclei and wild type genes in their macronuclei.
Note: The figures and legends in this page were contributed by Dr. Martin Gorovsky, Department of Biology, University of Rochester, Rochester, NY 14627. Phone: (716)275-6988. Fax: (716)275-2070. E-mail: email@example.com
Return to Top of this Page <<<<<<000>>>>>> Return to Main Page