Abstract
The cell wall–lacking mutant CW-15 of the unicellular green alga Chlamydomonas reinhardtii was transformed by electroporation using plasmid pCTVHyg, which was constructed with the hygromycin phosphotransferase genehpt as the selective marker and the Tn5 transposon of Escherichia coli under the control of the virus SV40 early gene promoter. Under optimal conditions (106 mid-exponential cells/ml; electric field strength 1 kV/cm; and pulse length 2 ms), the transformation yielded 103 HygR transformants per 106 recipient cells. The exogenous DNA integrated into the nuclear genome of Ch. reinhardtii was persistently inherited through more than 350 cell generations. The advantages of this system for the transformation ofCh. reinhardtii with heterologous genes are discussed.
Similar content being viewed by others
REFERENCES
Mayfield, S.P. and Kindle, K.L., Stable Nuclear Transformation of Chlamydomonas reinhardtii by Using a Chlamydomonas reinhardtii Gene as the Selectable Marker, Proc. Natl. Acad. Sci. USA, 1990, vol. 87, pp. 2087-2091.
Hall, L.M., Taylor, K.B., and Jones, D.D., Expression of a Foreign Gene in Chlamydomonas reinhardtii, Gene, 1993, vol. 124, pp. 75-81.
Rochaix, J.D. and van Dillewijn, J., Transformation of the Green Alga Chlamydomonas reinhardtii with Yeast DNA, Nature (London), 1982, vol. 296, pp. 70-72.
Boynton, I.E., Gillham, N.W., Harris, E.H., Newman, S.M., Randolf-Anderson, B.L., Jonson, A.M., and Jones, A.R., Chloroplast Transformation in Chlamydomonas with High-Velocity Microprojectiles, Science, 1998, vol. 240, pp. 1534-1538.
Kindle, K.L., High-Frequency Nuclear Transformation of Chlamydomonas reinhardtii, Proc. Natl. Acad. Sci. USA, 1990, vol. 87, pp. 1228-1232.
Newman, S.M., Boynton, J.E., Gillham, N.W., Randolph-Anderson, B.L., Johnson, A.M., and Harris, E.Y., Transformation of Chloroplast Ribosomal RNA Genes in Chlamydomonas: Molecular and Genetic Characterization of Integration Events, Genetics, 1990, vol. 126, pp. 875-888.
Bingham, S.E., Xu, R.H., and Webber, A.N., Transformation of Chloroplasts with the psaB Gene Encoding a Polypeptide of the Photosystem-I Reaction Center, FEBS Lett., 1991, vol. 292, pp. 137-140.
Debuchy, R., Rochaix, J.D., and Purton, S., The Argininosuccinate Lyase Gene of Chlamydomonas reinhardtii Is an Important Tool for Nuclear Transformation and for Correlating the Genetic and Molecular Maps of the arg7 Locus, EMBO J., 1989, vol. 8, pp. 2803-2809.
Sakamoto, W., Kindle, K.L., and Stern, D.B., In Vivo Analysis of the Chlamydomonas Chloroplast petD Gene Expression Using Stable Transformation of β-Glucuronidase Translation Fusions, Proc. Natl. Acad. Sci. USA, 1993, vol. 90, pp. 497-501.
Goldschmidt-Clermont, M., Transgenic Expression of Aminoglycoside Adenine Transferase in the Chloroplast: A Selectable Marker for Site-Directed Transformation of Chlamydomonas, Nucleic Acids Res., 1991, vol. 19, pp. 4083-4089.
Butanaev, A.M. and Ladygin, V.G., Soviet-Indian Symposium on Regulation of Photosynthesis, Pushchino, 1990, pp. 8-9.
Ladygin, V.G., The Pigment Mutants of Chlamydomonas reinhardtii Dang. Induced by N-Nitroso-N-Ethylurea and UV Light, Genetika, 1970, vol. 6,no. 3, pp. 42-50.
Plant Genetic Transformation and Gene Expression, Draper, J. et al., Eds., Oxford: Blackwell, 1988. Translated under the title Gennaya inzheneriya rastenii, Moscow: Mir, 1991, pp. 287–295.
Reiss, B., Sprengel, R., Will, H., and Schaller, H., A New Sensitive Method for Quantitative and Qualitative Assay of Neomycin Phosphotransferase in Crude Cell Extracts, Gene, 1984, vol. 30, pp. 211-218.
Gritz, L. and Davies, J., Plasmid-encoded Hygromycin B Resistance: The Sequence of Hygromycin B Phosphotransferase Gene and Its Expression in Escherichia coli and Saccharomyces cerevisiae, Gene, 1983, vol. 25, pp. 179-188.
Beck, E., Ludwig, D., Aurswald, E., Reiss, B., and Schaller, H., Nucleotide Sequence and Exact Localization of the Neomycin Phosphotransferase Gene from Transposon Tn5, Gene, 1982, vol. 19, pp. 327-336.
Maniatis, T., Fritsch, E.F., and Sambrook, J., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor: Cold Spring Harbor Lab., 1982. Translated under the title Molekulyarnoe klonirovanie, Moscow: Mir, 1984.
Tyurin, M.V. and Livshits, V.A., Electrotransformation of Bacteria: Problems and Prospects, Usp. Sovrem. Biol., 1993, vol. 113,no. 6, pp. 659-674.
Brown, L.E., Sprecher, S.L., and Keller, L.R., Introduction of Exogenous DNA into Chlamydomonas reinhardtii by Electroporation, Mol. Cell. Biol., 1991, vol. 11, pp. 2328-2332.
Rochaix, J.-D., van Dillewijn, J., and Rahire, M., Construction and Characterization of Autonomously Replicating Plasmids in the Green Unicellular Alga Chlamydomonas reinhardtii, Cell (Cambridge, Mass.), 1984, vol. 36, pp. 925-931.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Ladygin, V.G. The Transformation of the Unicellular Alga Chlamydomonas reinhardtii by Electroporation. Microbiology 72, 585–591 (2003). https://doi.org/10.1023/A:1026051402366
Issue Date:
DOI: https://doi.org/10.1023/A:1026051402366