Introduction of DNA into S. pombe Cells

Susan L. Forsburg1

1 The Salk Institute for Biological Studies, La Jolla, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 13.17
DOI:  10.1002/0471142727.mb1317s64
Online Posting Date:  November, 2003
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Abstract

Methods of transformation rely upon conditioning cells to take up DNA, and growing them under selective conditions to establish and maintain the plasmid or integration. Different methods may be used, including electroporation, treatment with lithium cations, or protoplast treatment which removes the cell wall. Protoplasts prepared as for transformation can also be induced to fuse with each other and undergo karyogamy, which provides a means of mating sterile strains.

     
 
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Table of Contents

  • Basic Protocol 1: Transformation Using Lithium Acetate
  • Alternate Protocol 1: Rapid Lithium Acetate Procedure
  • Alternate Protocol 2: Electroporation of S. Pombe Cells
  • Alternate Protocol 3: Protoplast Procedure
  • Support Protocol 1: Protoplast Fusion
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Transformation Using Lithium Acetate

  Materials
  • YES medium (unit 13.15)
  • S. pombe
  • MB medium (see recipe)
  • 0.1 M lithium acetate (adjust to pH 4.9 with acetic acid)
  • Plasmid DNA
  • TE, pH 7.5 ( appendix 22)
  • 50% (w/v) PEG 4000, 25° or 30°C
  • 50% YE medium (see recipe for YES in unit 13.15)
  • Selective EMM plates (units 13.15)
  • 25° or 32°C incubators
  • 1‐liter flask
  • 43°C water bath
  • Additional reagents and equipment for culturing yeast (unit 13.7)
NOTE: If necessary, a 30° rather than a 32°C incubator can be used.

Alternate Protocol 1: Rapid Lithium Acetate Procedure

  • Low‐glucose EMM with appropriate supplements (unit 13.15)
  • 100 mM lithium acetate/1mM EDTA (pH 4.9)
  • 40% (w/v) PEG 3350/100 mM lithium acetate/1mM EDTA, pH 4.9
  • 42°C water bath
  • Additional reagents and equipment for growth of yeast (unit 13.7)

Alternate Protocol 2: Electroporation of S. Pombe Cells

  • EMM medium (unit 13.15)
  • Cit/phos/EDTA (see recipe)
  • Cit/phos/sorbitol (see recipe)
  • NovoZym 234 (BiosPacific)
  • 10 mM Tris⋅Cl, pH 7.6 ( appendix 22)/1.2 M sorbitol
  • 10 mM Tris⋅Cl, pH 7.6 ( appendix 22)/10 mM CaCl 2/1.2 M sorbitol
  • 10 mM Tris⋅Cl, pH 7.6 ( appendix 22)/10 mM CaCl 2/20% (w/v) PEG 4000
  • CaCl 2/sorb/YE (see recipe)
  • Selective EMM sorbitol plates (unit 13.15)
  • 50‐ml plastic centrifuge tube
  • 37°C incubator
  • 29° to 32°C incubator
  • Additional reagents and equipment for counting cells using a hemacytometer ( appendix 3F)

Alternate Protocol 3: Protoplast Procedure

  • Strains with complementary markers
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Figures

Videos

Literature Cited

Literature Cited
   Alfa, C., Fantes, P., Hyams, J., McLeod, M., and Warbrick, E. 1993. Experiments With Fission Yeast. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Bähler, J., Wu, J.Q., Longtine, M.S., Shah, N.G., McKenzie, A., Steever, A.B., Wach, A., Philippsen, P., and Pringle, J.R. 1998. Heterologous modules for efficient and versatile PCR‐based gene targeting in Schizosaccharomyces pombe. Yeast 14:943‐951.
   Beach, D. and Nurse, P. 1981. High frequency transformation of the fission yeast S. pombe. Nature 200:140‐142.
   Broker, M. 1993. Rapid transformation of cryopreserved competent Schizosaccharomyces pombe cells. Biotechniques 15:598.
   Gould, K.L., ed. 2003. Methods. Academic Press, San Diego.
   Grallert, B., Nurse, P., and Patterson, T.E. 1993. A study of integrative transformation in Schizosaccharomyces pombe. Mol. Gen. Genet. 238:26‐32.
   Jimenez, J. 1991. Cryopreservation of competent Schizosaccharomyces pombe protoplasts. Trends Genet. 7:40.
   Kanter‐smoler, G., Dahlkvist, A., and Sunnerhagen, P. 1994. Improved method for rapid transformation of intact Schizosaccharomyces pombe cells. Biotechniques 16:798.
   Keeney, J.B. and Boeke, J.D. 1994. Efficient targeted integration at leu1‐32 and ura4‐294 in Schizosaccharomyces pombe. Genetics 136:849‐856.
   Moreno, S., Klar, A., and Nurse, P. 1991. Molecular genetic analysis of the fission yeast Schizosaccharomyces pombe. Meth. Enzymol. 194:795‐823.
   Okazaki, K., Okazaki, N., Kume, K., Jinno, S., Tanaka, K., and Okayama, H. 1990. High‐frequency transformation method and library transducing vectors for cloning mammalian cDNAs by trans‐complementation of Schizosaccharomyces pombe. Nucl. Acids Res. 18:6485‐6489.
   Russell, P. 1989. Gene cloning and expression in fission yeast. In Molecular Biology of the Fission Yeast (A. Nasim, P.G. Young, and, B.F. Johnson, eds.) pp. 243‐271. Academic Press, San Diego.
   Siam, R., Dolan, W.P., and Forsburg, S.L. 2003. Choosing and using S. pombe plasmids. Methods In Press.
   Suga, M. and Hatakeyama, T. 2001. High efficiency transformation of Schizosaccharomyces pombe pretreated with thiol compounds by electroporation. Yeast 18:1015‐1021.
   Suga, M. and Hatakeyama, T. 2003. High‐efficiency electroporation by freezing intact yeast cells with addition of calcium. Curr. Genet. 43:206‐211.
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