Abstract
A method for introducing heterologous DNA into Saccharomyces cerevisiae rapidly and efficiently by electroporation was developed. Transformant colonies appeared somewhat sooner than by the LiCl or spheroplast transformation method, and the time spent in manipulation was much less than for these two methods. The pores in the cell membrane formed by the high voltage of electroporation were resealed within 6 to 7 min after electroporation. At a capacitance of 25 microF, the optimum voltage was 2.0 to 2.25 kV/cm. Log-phase cells concentrated to 10 to 20 units at an optical density of 600 nm in 200 microliters of fresh rich medium and electroporated at 2.25 kV/cm in the presence of 0.1 microgram of supercoiled plasmid DNA will yield 1,000 to 4,500 colonies per microgram of DNA.
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- Beggs J. D. Transformation of yeast by a replicating hybrid plasmid. Nature. 1978 Sep 14;275(5676):104–109. doi: 10.1038/275104a0. [DOI] [PubMed] [Google Scholar]
- Bitter G. A., Egan K. M., Koski R. A., Jones M. O., Elliott S. G., Giffin J. C. Expression and secretion vectors for yeast. Methods Enzymol. 1987;153:516–544. doi: 10.1016/0076-6879(87)53076-2. [DOI] [PubMed] [Google Scholar]
- Brzobohatý B., Kovác L. Factors enhancing genetic transformation of intact yeast cells modify cell wall porosity. J Gen Microbiol. 1986 Nov;132(11):3089–3093. doi: 10.1099/00221287-132-11-3089. [DOI] [PubMed] [Google Scholar]
- Clarke L., Carbon J. Isolation of a yeast centromere and construction of functional small circular chromosomes. Nature. 1980 Oct 9;287(5782):504–509. doi: 10.1038/287504a0. [DOI] [PubMed] [Google Scholar]
- Elliott S., Giffin J., Suggs S., Lau E. P., Banks A. R. Secretion of glycosylated human erythropoietin from yeast directed by the alpha-factor leader region. Gene. 1989 Jun 30;79(1):167–180. doi: 10.1016/0378-1119(89)90102-9. [DOI] [PubMed] [Google Scholar]
- Fitzgerald-Hayes M., Clarke L., Carbon J. Nucleotide sequence comparisons and functional analysis of yeast centromere DNAs. Cell. 1982 May;29(1):235–244. doi: 10.1016/0092-8674(82)90108-8. [DOI] [PubMed] [Google Scholar]
- Hinnen A., Hicks J. B., Fink G. R. Transformation of yeast. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1929–1933. doi: 10.1073/pnas.75.4.1929. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Klebe R. J., Harriss J. V., Sharp Z. D., Douglas M. G. A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast. Gene. 1983 Nov;25(2-3):333–341. doi: 10.1016/0378-1119(83)90238-x. [DOI] [PubMed] [Google Scholar]
- Neumann E., Schaefer-Ridder M., Wang Y., Hofschneider P. H. Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J. 1982;1(7):841–845. doi: 10.1002/j.1460-2075.1982.tb01257.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Riemann F., Zimmermann U., Pilwat G. Release and uptake of haemoglobin and ions in red blood cells induced by dielectric breakdown. Biochim Biophys Acta. 1975 Jul 3;394(3):449–462. doi: 10.1016/0005-2736(75)90296-5. [DOI] [PubMed] [Google Scholar]
- Struhl K., Stinchcomb D. T., Scherer S., Davis R. W. High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1035–1039. doi: 10.1073/pnas.76.3.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Uno I., Fukami K., Kato H., Takenawa T., Ishikawa T. Essential role for phosphatidylinositol 4,5-bisphosphate in yeast cell proliferation. Nature. 1988 May 12;333(6169):188–190. doi: 10.1038/333188a0. [DOI] [PubMed] [Google Scholar]
- Vienken J., Jeltsch E., Zimmermann U. Penetration and entrapment of large particles in erythrocytes by electrical breakdown techniques. Cytobiologie. 1978 Jun;17(1):182–196. [PubMed] [Google Scholar]
- Zimmermann U., Pilwat G., Riemann F. Preparation of erythrocyte ghosts by dielectric breakdown of the cell membrane. Biochim Biophys Acta. 1975 Jan 28;375(2):209–219. doi: 10.1016/0005-2736(75)90189-3. [DOI] [PubMed] [Google Scholar]
- Zlotnik H., Fernandez M. P., Bowers B., Cabib E. Saccharomyces cerevisiae mannoproteins form an external cell wall layer that determines wall porosity. J Bacteriol. 1984 Sep;159(3):1018–1026. doi: 10.1128/jb.159.3.1018-1026.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]