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. 1998 Apr;148(4):1821–1828. doi: 10.1093/genetics/148.4.1821

High-efficiency transformation of Chlamydomonas reinhardtii by electroporation.

K Shimogawara 1, S Fujiwara 1, A Grossman 1, H Usuda 1
PMCID: PMC1460073  PMID: 9560396

Abstract

We have established a high-efficiency method for transforming the unicellular, green alga Chlamydomonas reinhardtii by electroporation. Electroporation of strains CC3395 and CC425, cell wall-less mutants devoid of argininosuccinate lyase (encoded by ARG7), in the presence of the plasmid pJD67 (which contains ARG7) was used to optimize conditions for the introduction of exogenous DNA. The conditions that were varied included osmolarity, temperature, concentration of exogenous DNA, voltage and capacitance. Following optimization, the maximum transformation frequency obtained was 2 x 10(5) transformants per microg of DNA; this frequency is two orders of magnitude higher than obtained with the current standard method using glass beads to introduce exogenous DNA. The electroporation procedure described in this article is of general utility, and makes it feasible to isolate genes by direct complementation of Chlamydomonas reinhardtii mutants.

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Selected References

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  1. Boynton J. E., Gillham N. W., Harris E. H., Hosler J. P., Johnson A. M., Jones A. R., Randolph-Anderson B. L., Robertson D., Klein T. M., Shark K. B. Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science. 1988 Jun 10;240(4858):1534–1538. doi: 10.1126/science.2897716. [DOI] [PubMed] [Google Scholar]
  2. Brown L. E., Sprecher S. L., Keller L. R. Introduction of exogenous DNA into Chlamydomonas reinhardtii by electroporation. Mol Cell Biol. 1991 Apr;11(4):2328–2332. doi: 10.1128/mcb.11.4.2328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Costanzo M. C., Fox T. D. Transformation of yeast by agitation with glass beads. Genetics. 1988 Nov;120(3):667–670. doi: 10.1093/genetics/120.3.667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Davies J. P., Grossman A. R. Sequences controlling transcription of the Chlamydomonas reinhardtii beta 2-tubulin gene after deflagellation and during the cell cycle. Mol Cell Biol. 1994 Aug;14(8):5165–5174. doi: 10.1128/mcb.14.8.5165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Davies J. P., Weeks D. P., Grossman A. R. Expression of the arylsulfatase gene from the beta 2-tubulin promoter in Chlamydomonas reinhardtii. Nucleic Acids Res. 1992 Jun 25;20(12):2959–2965. doi: 10.1093/nar/20.12.2959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Davies J. P., Yildiz F., Grossman A. R. Mutants of Chlamydomonas with Aberrant Responses to Sulfur Deprivation. Plant Cell. 1994 Jan;6(1):53–63. doi: 10.1105/tpc.6.1.53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dunahay T. G. Transformation of Chlamydomonas reinhardtii with silicon carbide whiskers. Biotechniques. 1993 Sep;15(3):452-5, 457-8, 460. [PubMed] [Google Scholar]
  8. Funke R. P., Kovar J. L., Weeks D. P. Intracellular carbonic anhydrase is essential to photosynthesis in Chlamydomonas reinhardtii at atmospheric levels of CO2. Demonstration via genomic complementation of the high-CO2-requiring mutant ca-1. Plant Physiol. 1997 May;114(1):237–244. doi: 10.1104/pp.114.1.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gietz R. D., Schiestl R. H., Willems A. R., Woods R. A. Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast. 1995 Apr 15;11(4):355–360. doi: 10.1002/yea.320110408. [DOI] [PubMed] [Google Scholar]
  10. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  11. Infante A., Lo S., Hall J. L. A Chlamydomonas genomic library in yeast artificial chromosomes. Genetics. 1995 Sep;141(1):87–93. doi: 10.1093/genetics/141.1.87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kindle K. L. High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1228–1232. doi: 10.1073/pnas.87.3.1228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kindle K. L., Schnell R. A., Fernández E., Lefebvre P. A. Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase. J Cell Biol. 1989 Dec;109(6 Pt 1):2589–2601. doi: 10.1083/jcb.109.6.2589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Manivasakam P., Schiestl R. H. High efficiency transformation of Saccharomyces cerevisiae by electroporation. Nucleic Acids Res. 1993 Sep 11;21(18):4414–4415. doi: 10.1093/nar/21.18.4414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Newman S. M., Gillham N. W., Harris E. H., Johnson A. M., Boynton J. E. Targeted disruption of chloroplast genes in Chlamydomonas reinhardtii. Mol Gen Genet. 1991 Nov;230(1-2):65–74. doi: 10.1007/BF00290652. [DOI] [PubMed] [Google Scholar]
  16. Purton S., Rochaix J. D. Complementation of a Chlamydomonas reinhardtii mutant using a genomic cosmid library. Plant Mol Biol. 1994 Feb;24(3):533–537. doi: 10.1007/BF00024121. [DOI] [PubMed] [Google Scholar]
  17. Quinn J. M., Merchant S. Two copper-responsive elements associated with the Chlamydomonas Cyc6 gene function as targets for transcriptional activators. Plant Cell. 1995 May;7(5):623–628. doi: 10.1105/tpc.7.5.623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Randolph-Anderson B. L., Boynton J. E., Gillham N. W., Harris E. H., Johnson A. M., Dorthu M. P., Matagne R. F. Further characterization of the respiratory deficient dum-1 mutation of Chlamydomonas reinhardtii and its use as a recipient for mitochondrial transformation. Mol Gen Genet. 1993 Jan;236(2-3):235–244. doi: 10.1007/BF00277118. [DOI] [PubMed] [Google Scholar]
  19. Rochaix J. D. Chlamydomonas reinhardtii as the photosynthetic yeast. Annu Rev Genet. 1995;29:209–230. doi: 10.1146/annurev.ge.29.120195.001233. [DOI] [PubMed] [Google Scholar]
  20. Schnell R. A., Lefebvre P. A. Isolation of the Chlamydomonas regulatory gene NIT2 by transposon tagging. Genetics. 1993 Jul;134(3):737–747. doi: 10.1093/genetics/134.3.737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Tam L. W., Lefebvre P. A. Cloning of flagellar genes in Chlamydomonas reinhardtii by DNA insertional mutagenesis. Genetics. 1993 Oct;135(2):375–384. doi: 10.1093/genetics/135.2.375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Tang D. K., Qiao S. Y., Wu M. Insertion mutagenesis of Chlamydomonas reinhardtii by electroporation and heterologous DNA. Biochem Mol Biol Int. 1995 Aug;36(5):1025–1035. [PubMed] [Google Scholar]
  23. Vashishtha M., Segil G., Hall J. L. Direct complementation of Chlamydomonas mutants with amplified YAC DNA. Genomics. 1996 Sep 15;36(3):459–467. doi: 10.1006/geno.1996.0491. [DOI] [PubMed] [Google Scholar]
  24. Zhang H., Herman P. L., Weeks D. P. Gene isolation through genomic complementation using an indexed library of Chlamydomonas reinhardtii DNA. Plant Mol Biol. 1994 Feb;24(4):663–672. doi: 10.1007/BF00023562. [DOI] [PubMed] [Google Scholar]

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