DOI: 10.1093/genetics/148.4.1821. High-Efficiency Transformation of Chlamydomonas reinhardtii by Electroporation

High-Efficiency Transformation of Chlamydomonas reinhardtii by Electroporation

10.1093/genetics/148.4.1821

Crossref journal-article

Oxford University Press (OUP)

Genetics (286)

Abstract

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 × 105 transformants per μg 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.

Bibliography

Shimogawara, K., Fujiwara, S., Grossman, A., & Usuda, H. (1998). High-Efficiency Transformation of Chlamydomonas reinhardtii by Electroporation. Genetics, 148(4), 1821â1828.

Authors 4

Kosuke Shimogawara (first)
Shoko Fujiwara (additional)
Arthur Grossman (additional)
Hideaki Usuda (additional)

References 30 Referenced 386

10.1126/science.2897716 / Science / Chloroplast transformation in Chlamydomonas with high-velocity microprojectiles by Boynton (1988)
{'key': '2022010308592291000_R2', 'first-page': '2328', 'article-title': 'Introduction of exogenous DNA into Chlamydomonas reinhardtii by electroporation', 'volume': '11', 'author': 'Brown', 'year': '1991', 'journal-title': 'Mol. Cell. Biol.'} / Mol. Cell. Biol. / Introduction of exogenous DNA into Chlamydomonas reinhardtii by electroporation by Brown (1991)
{'key': '2022010308592291000_R3', 'first-page': '682', 'article-title': 'Hygromycin phosphotransferase gene as a donimant selective marker for transformation of Chlamydomonas renhardtii', 'volume': '28', 'author': 'Butanaev', 'year': '1994', 'journal-title': 'Mol. Biol.'} / Mol. Biol. / Hygromycin phosphotransferase gene as a donimant selective marker for transformation of Chlamydomonas renhardtii by Butanaev (1994)
10.1093/genetics/120.3.667 / Genetics / Transformation of yeast by agitation with glass beads by Costanzo (1988)
{'key': '2022010308592291000_R5', 'first-page': '5165', 'article-title': 'Sequences controlling transcription of the Chlamydomonas renhardtii β2-tubulin gene after deflagellation and during the cell cycle', 'volume': '14', 'author': 'Davies', 'year': '1994', 'journal-title': 'Mol. Cell. Biol.'} / Mol. Cell. Biol. / Sequences controlling transcription of the Chlamydomonas renhardtii β2-tubulin gene after deflagellation and during the cell cycle by Davies (1994)
10.1093/nar/20.12.2959 / Nucleic Acids Res. / Expression of the arylsulfatase gene from the β2-tubulin promotor in Chlamydomonas reinhardtii by Davies (1992)
10.2307/3869674 / Plant Cell / Mutants of Chlamydomonas with aberrant responses to sulfur deprivation by Davies (1994)
10.1002/j.1460-2075.1996.tb00568.x / EMBO J. / SacI, a putative regulator that is critical for survival of Chlamydomonas reinhardtii during sulfur deprivation by Davies (1996)
{'key': '2022010308592291000_R9', 'first-page': '452', 'article-title': 'Transformation of Chlamydomonas reinhardtii with silicon carbide whiskers', 'volume': '15', 'author': 'Dunahay', 'year': '1993', 'journal-title': 'BioTechniques'} / BioTechniques / Transformation of Chlamydomonas reinhardtii with silicon carbide whiskers by Dunahay (1993)
10.1104/pp.114.1.237 / Plant Physiol. / Intracellular carbonic anhydrase is essential to photosynthesis in Chlamydomonas reinhardtii at atmospheric levels of CO2 by Funke (1997)
10.1002/yea.320110408 / Yeast / Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure by Gietz (1995)
10.1016/S0022-2836(83)80284-8 / J. Mol. Biol. / Studies on transformation of Escherichia coli with plasmids by Hanahan (1983)
{'volume-title': 'The Chlamydomonas Sourcebook', 'year': '1989', 'author': 'Harris', 'key': '2022010308592291000_R13'} / The Chlamydomonas Sourcebook by Harris (1989)
10.1093/genetics/141.1.87 / Genetics / A Chlamydomonas genomic library in yeast artificial chromosomes by Infante (1995)
10.1073/pnas.87.3.1228 / Proc. Natl. Acad. Sci. USA / High-frequency nuclear transformation of Chlamydomonas reinhardtii by Kindle (1990)
10.1083/jcb.109.6.2589 / J. Cell Biol. / Stable transformation of Chlamydomonas using Chlamydomonas gene for nitrate reductase by Kindle (1989)
10.1093/nar/21.18.4414 / Nucleic Acids Res. / High efficiency transformation of Saccharomyces cerevisiae by electroporation by Manivasakam (1993)
10.1007/BF00290652 / Mol. Gen. Genet. / Targeted disruption of chloroplast genes in Chlamydomonas reinhardtii by Newman (1991)
10.1007/BF00024121 / Plant Mol. Biol. / Complementation of a Chlamydomonas reinhardtii mutant using a genomic cosmid library by Purton (1994)
{'key': '2022010308592291000_R20', 'first-page': '623', 'article-title': 'Two copper-responsive elements associated with the Chlamydomonas Cys6 gene function as targets for transcriptional activators', 'volume': '7', 'author': 'Quinn', 'year': '1995', 'journal-title': 'Plant Cell'} / Plant Cell / Two copper-responsive elements associated with the Chlamydomonas Cys6 gene function as targets for transcriptional activators by Quinn (1995)
10.1007/BF00277118 / Mol. Gen. Genet. / Further characterization of the respiratory deficient dum-1 mutation of Chlamydomonas reinhardtii and its use as a recipient for mitochondrial transformation by Randolph-Anderson (1993)
10.1146/annurev.ge.29.120195.001233 / Annu. Rev. Genet. / Chlamydomonas reinardtii as the photosynthetic yeast by Rochaix (1995)
{'key': '2022010308592291000_R23', 'volume-title': 'Molecular Cloning', 'author': 'Sambrook', 'year': '1989', 'edition': 'Ed. 2.'} / Molecular Cloning by Sambrook (1989)
10.1007/BF00340712 / Curr. Genet. / High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier by Schiestl (1989)
10.1093/genetics/134.3.737 / Genetics / Isolation of the Chlamydomonas regulatory gene NIT2 by transposon tagging by Schnell (1993)
10.1073/pnas.90.19.9199 / Proc. Natl. Acad. Sci. USA / Homologous recombination in the nuclear genome of Chlamydomonas reinhardtii by Sodeinde (1993)
10.1093/genetics/135.2.375 / Genetics / Cloning of flagella genes in Chlamydomonas reinhardtii by DNA insertional mutagenesis by Tam (1993)
{'key': '2022010308592291000_R28', 'first-page': '1025', 'article-title': 'Insertion mutagenesis of Chlamydomonas reinhardtii by electroporation and heterologous DNA', 'volume': '36', 'author': 'Tang', 'year': '1995', 'journal-title': 'Biochem. Mol. Biol. Int.'} / Biochem. Mol. Biol. Int. / Insertion mutagenesis of Chlamydomonas reinhardtii by electroporation and heterologous DNA by Tang (1995)
10.1006/geno.1996.0491 / Genomics / Direct complementation of Chlamydomonas mutants with amplified YAC DNA by Vashishtha (1996)
10.1007/BF00023562 / Plant Mol. Biol. / Gene isolation through genomic complementation using an indexed cosmid library of Chlamydomonas reinhardtii DNA by Zhang (1994)

Dates

Type	When
Created	4 years, 6 months ago (Feb. 12, 2021, 6:52 p.m.)
Deposited	3 years, 8 months ago (Jan. 3, 2022, 4 a.m.)
Indexed	1 day, 15 hours ago (Sept. 3, 2025, 6:52 a.m.)
Issued	27 years, 5 months ago (April 1, 1998)
Published	27 years, 5 months ago (April 1, 1998)
Published Online	27 years, 5 months ago (April 1, 1998)
Published Print	27 years, 5 months ago (April 1, 1998)

Funders 0

None

BibTeX

@article{Shimogawara_1998, title={High-Efficiency Transformation of Chlamydomonas reinhardtii by Electroporation}, volume={148}, ISSN={1943-2631}, url={http://dx.doi.org/10.1093/genetics/148.4.1821}, DOI={10.1093/genetics/148.4.1821}, number={4}, journal={Genetics}, publisher={Oxford University Press (OUP)}, author={Shimogawara, Kosuke and Fujiwara, Shoko and Grossman, Arthur and Usuda, Hideaki}, year={1998}, month=apr, pages={1821–1828} }

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  "references-count": 30,
  "journal-issue": {
    "issue": "4",
    "published-online": {
      "date-parts": [
        [
          1998,
          4,
          1
        ]
      ]
    },
    "published-print": {
      "date-parts": [
        [
          1998,
          4,
          1
        ]
      ]
    }
  },
  "URL": "http://dx.doi.org/10.1093/genetics/148.4.1821",
  "relation": {},
  "ISSN": [
    "1943-2631"
  ],
  "subject": [],
  "published-other": {
    "date-parts": [
      [
        1998,
        4,
        1
      ]
    ]
  },
  "published": {
    "date-parts": [
      [
        1998,
        4,
        1
      ]
    ]
  }
}