Efficient Mass Transformation of Tetrahymena thermophila by Electroporation of Conjugants

Conjugating cells of the ciliate Tetrahymena thermophila were electroporated in the presence of plasmid DNA containing a paromomycin-resistant ribosomal RNA gene (rDNA). Cells were selected with paromomycin following 12-24 hr of growth on nonselective medium. Resistant cells appeared after 2-3 days....

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1992-10, Vol.89 (19), p.9196-9200
Main Authors: Gaertig, Jacek, Gorovsky, Martin A.
Format: Article
Language:English
Subjects:
Animals
Biological and medical sciences
Biotechnology
Blotting, Southern
Cell growth
Conjugation, Genetic
Deoxyribonucleic acid
DNA
DNA, Ribosomal - genetics
DNA, Ribosomal - isolation & purification
Drug Resistance - genetics
Electric Stimulation
Electroporation
Fundamental and applied biological sciences. Psychology
Genetic engineering
Genetic technics
Genetic Techniques
Genetic Vectors
Genetics
Macronucleus
Methods. Procedures. Technologies
Microinjections
Miscellaneous
Paromomycin - pharmacology
Plasmids
Replication origin
Ribonucleic acid
Ribosomal DNA
RNA
Somatic cells
Synthetic digonucleotides and genes. Sequencing
Tetrahymena thermophila
Tetrahymena thermophila - drug effects
Tetrahymena thermophila - genetics
Transfection
Transformation, Genetic
Yeast
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Summary:Conjugating cells of the ciliate Tetrahymena thermophila were electroporated in the presence of plasmid DNA containing a paromomycin-resistant ribosomal RNA gene (rDNA). Cells were selected with paromomycin following 12-24 hr of growth on nonselective medium. Resistant cells appeared after 2-3 days. Processing vectors containing the micronuclear rDNA and somatic vectors containing the macronuclear gene transformed the cells, with the former yielding frequencies up to 900 transformants per μ g of plasmid DNA. A ribosomal protein gene (rpL29) conferring cycloheximide resistance also transformed conjugating cells. The transformation efficiency of the plasmid containing only the rpL29 gene was increased by insertion of an rDNA replication origin and by cotransformation and preselection with an rDNA vector. These results indicate that electroporation can be used for the production of large numbers of transformed Tetrahymena.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.89.19.9196