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Genetic Transformation of the Model Green Alga Chlamydomonas reinhardtii

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Transgenic Plants

Part of the book series: Methods in Molecular Biology ((MIMB,volume 847))

Abstract

Over the past three decades, the single-celled green alga Chlamydomonas reinhardtii has become an invaluable model organism in plant biology and an attractive production host in biotechnology. The genetic transformation of Chlamydomonas is relatively simple and efficient, but achieving high expression levels of foreign genes has remained challenging. Here, we provide working protocols for algal cultivation and transformation as well as for selection and analysis of transgenic algal clones. We focus on two commonly used transformation methods for Chlamydomonas: glass bead-assisted transformation and particle gun-mediated (biolistic) transformation. In addition, we describe available tools for promoting efficient transgene expression and highlight important considerations for designing transformation vectors.

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References

  1. Harris, E. H. (2001) Chlamydomonas as a model organism. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 363–406.

    Article  PubMed  CAS  Google Scholar 

  2. Pröschold, T., Harris, E. H., and Coleman, A. W. (2005) Portrait of a species: Chlamydomonas reinhardtii. Genetics 170, 1601–1610.

    Article  PubMed  Google Scholar 

  3. Hippler, M., Redding, K., and Rochaix, J.-D. (1998) Chlamydomonas genetics, a tool for the study of bioenergetic pathways. Biochim. Biophys. Acta 1367, 1–62.

    Article  PubMed  CAS  Google Scholar 

  4. Kindle, K. L., Richards, K. L., and Stern, D. B. (1991) Engineering the chloroplast genome: Techniques and capabilities for chloroplast transformation in Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA 88, 1721–1725.

    Article  PubMed  CAS  Google Scholar 

  5. Remacle, C., Cardol, P., Coosemans, N., Gaisne, M., and Bonnefoy, N. (2006) High-efficiency biolistic transformation of Chlamydomonas mitochondria can be used to insert mutations in complex I genes. Proc. Natl. Acad. Sci. USA 103, 4771–4776.

    Article  PubMed  CAS  Google Scholar 

  6. Kindle, K. L. (1990) High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA 87, 1228–1232.

    Article  PubMed  CAS  Google Scholar 

  7. Merchant, S. S., Prochnik, S. E., Vallon, O., Harris, E. H., Karpowicz, S. J., Witman, G. B., Terry, A., Salamov, A., Fritz-Laylin, L. K., Maréchal-Drouard, L., Marshall, W. F., Qu, L.-H., Nelson, D. R., Sanderfoot, A. A., Spalding, M. H., Kapitonov, V. V., Ren, Q., Ferris, P., Lindquist, E., Shapiro, H., Lucas, S. M., Grimwood, J., Schmutz, J., Chlamydomonas Annotation Team, JGI Annotation Team, Grigoriev, I. V., Rokhsar, D. S. and Grossman, A. R. (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318, 245–251.

    Article  PubMed  CAS  Google Scholar 

  8. Dent, R. M., Haglund, C. M., Chin, B. L., Kobayashi, M. C., and Niyogi, K. K. (2005) Functional genomics of eukaryotic photosynthesis using insertional mutagenesis of Chlamydomonas reinhardtii. Plant Physiol. 137, 545–556.

    Article  PubMed  CAS  Google Scholar 

  9. Rohr, J., Sarkar, N., Balenger, S., Jeong, B.-r, and Cerutti, H. (2004) Tandem inverted repeat system for selection of effective transgenic RNAi strains in Chlamydomonas. Plant J. 40, 611–621.

    Article  PubMed  CAS  Google Scholar 

  10. Molnar, A., Bassett, A., Thuenemann, E., Schwach, F., Karkare, S., Ossowski, S., Weigel, D., and Baulcombe, D. (2009) Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii. Plant J. 58, 165–174.

    Article  PubMed  CAS  Google Scholar 

  11. Zhao, T., Wang, W., Bai, X., and Qi, Y. (2009) Gene silencing by artificial micro RNAs in Chlamydomonas. Plant J. 58, 157–164.

    Article  PubMed  CAS  Google Scholar 

  12. Pedersen, L. B., Geimer, S., and Rosenbaum, J. L. (2006) Dissecting the molecular mechanisms of intraflagellar transport in Chlamydomonas. Curr. Biol. 16, 450–459.

    Article  PubMed  CAS  Google Scholar 

  13. Schmidt, M., Geßner, G., Luff, M., Heiland, I., Wagner, V., Kaminski, M., Geimer, S., Eitzinger, N., Reißenweber, T., Voytsekh, O., Fiedler, M., Mittag, M., and Kreimer, G. (2006) Proteomic analysis of the eyespot of Chlamydomonas reinhardtii provides novel insights into its components and tactic movements. Plant Cell 18, 1908–1930.

    Article  PubMed  CAS  Google Scholar 

  14. Walker, T. L., Purton, S., Becker, D. K., and Collet, C. (2005) Microalgae as bioreactors. Plant Cell Rep. 24, 629–641.

    Article  CAS  Google Scholar 

  15. Radakovits, R., Jinkerson, R. E., Darzins, A., and Posewitz, M. C. (2010) Genetic engineering of algae for enhanced biofuel production. Eukaryot. Cell 9, 486–501.

    Article  PubMed  CAS  Google Scholar 

  16. Fuhrmann, M., Oertel, W., and Hegemann, P. (1999) A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii. Plant J. 19, 353–361.

    Article  PubMed  CAS  Google Scholar 

  17. Schroda, M., Blöcker, D., and Beck, C. F. (2000) The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas. Plant J. 21, 121–131.

    Article  PubMed  CAS  Google Scholar 

  18. Harris, E. H. (1989) The Chlamydomonas Sourcebook. Academic Press, San Diego, CA. 780 pp.

    Google Scholar 

  19. Sizova, I., Fuhrmann, M., and Hegemann, P. (2001) A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii. Gene 277, 221–229.

    Article  PubMed  CAS  Google Scholar 

  20. Nelson, J. A. E., Savereide, P. B., and Lefebvre, P. A. (1994) The CRY1 gene in Chlamydomonas reinhardtii: Structure and use as a dominant selectable marker for nuclear transformation. Mol. Cell. Biol. 14, 4011–4019.

    PubMed  CAS  Google Scholar 

  21. Lumbreras, V., Stevens, D. R., and Purton, S. (1998) Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron. Plant J. 14, 441–447.

    Article  CAS  Google Scholar 

  22. Berthold, P., Schmitt, R., and Mages, W. (2002) An engineered Streptomyces hygroscopicus aph 7” gene mediates dominant resistance against hygromycin B in Chlamydomonas reinhardtii. Protist 153, 401–412.

    Article  PubMed  CAS  Google Scholar 

  23. Auchincloss, A. H., Loroch, A. I., and Rochaix, J.-D. (1999) The argininosuccinate lyase gene of Chlamydomonas reinhardtii: cloning of the cDNA and its characterization as a selectable shuttle marker. Mol. Gen. Genet. 261, 21–30.

    Article  PubMed  CAS  Google Scholar 

  24. Karcher, D., Köster, D., Schadach, A., Klevesath, A., and Bock, R. (2009) The Chlamydomonas chloroplast HLP protein is required for nucleoid organization and genome maintenance. Mol. Plant 2, 1223–1232.

    Article  PubMed  CAS  Google Scholar 

  25. Neupert, J., Karcher, D., and Bock, R. (2009) Generation of Chlamydomonas strains that efficiently express nuclear transgenes. Plant J. 57, 1140–1150.

    Article  PubMed  CAS  Google Scholar 

  26. Fuhrmann, M., Hausherr, A., Ferbitz, L., Schödl, T., Heitzer, M., and Hegemann, P. (2004) Monitoring dynamic expression of nuclear genes in Chlamydomonas reinhardtii by using a synthetic luciferase reporter gene. Plant Mol. Biol. 55, 869–881.

    PubMed  CAS  Google Scholar 

  27. Shao, N. and Bock, R. (2008) A codon-optimized luciferase from Gaussia princeps facilitates the in vivo monitoring of gene expression in the model alga Chlamydomonas reinhardtii. Curr. Genet. 53, 381–388.

    Article  PubMed  CAS  Google Scholar 

  28. Fischer, N. and Rochaix, J.-D. (2001) The flanking regions of PsaD drive efficient gene expression in the nucleus of the green alga Chlamydomonas reinhardtii. Mol. Genet. Genom. 265, 888–894.

    Article  CAS  Google Scholar 

  29. Klein, U., Chen, C., Gibbs, M. and Platt-Aloia, K. A. (1983) Cellular fractionation of Chlamydomonas reinhardtii with emphasis on the isolation of the chloroplast. Plant Physiol. 72, 481–487.

    Article  PubMed  CAS  Google Scholar 

  30. Bock, R. (2004) Studying RNA editing in transgenic chloroplasts of higher plants. Methods Mol. Biol. 265, 345–356.

    PubMed  CAS  Google Scholar 

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Acknowledgments

Work on Chlamydomonas in the authors’ laboratory is supported by a grant in the Systems Biology Program (FORSYS) from the Bundesministerium für Bildung und Forschung (BMBF) and by the Max Planck Society.

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Correspondence to Ralph Bock .

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Neupert, J., Shao, N., Lu, Y., Bock, R. (2012). Genetic Transformation of the Model Green Alga Chlamydomonas reinhardtii . In: Dunwell, J., Wetten, A. (eds) Transgenic Plants. Methods in Molecular Biology, vol 847. Humana Press. https://doi.org/10.1007/978-1-61779-558-9_4

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  • DOI: https://doi.org/10.1007/978-1-61779-558-9_4

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-557-2

  • Online ISBN: 978-1-61779-558-9

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