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Double targeted gene replacement for creating null mutants.

A Cruz1, C M Coburn, S M Beverley

  • 1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.

Proceedings of the National Academy of Sciences of the United States of America
|August 15, 1991
PubMed
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Researchers developed a double gene targeting method to create homozygous gene replacements in Leishmania major. This technique enables functional genetic studies in asexual diploids and may lead to improved vaccines for leishmaniasis.

Area of Science:

  • Parasitology
  • Molecular Biology
  • Genetics

Background:

  • Leishmania major is an asexual diploid protozoan parasite.
  • Existing gene targeting methods have limitations for creating homozygous gene replacements.
  • Functional genetic analysis in asexual diploids requires robust gene replacement strategies.

Purpose of the Study:

  • To develop and validate a double gene targeting method for creating homozygous gene replacements in Leishmania major.
  • To introduce and assess an improved hygromycin B-resistance cassette (HYG) as a selectable marker.
  • To demonstrate the utility of the method for creating auxotrophic mutants and its potential applications.

Main Methods:

  • Utilized double gene targeting with two independent selectable markers (hygromycin phosphotransferase (HYG) and neomycin phosphotransferase (NEO)).

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  • Developed and tested HYG-containing vectors for Leishmania transformation and gene targeting.
  • Performed gene replacement of the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene in a heterozygous Leishmania line.

    • Main Results:

    • The HYG marker functioned equivalently to the previously used NEO marker.
    • Drug resistances conferred by NEO and HYG were independent, allowing simultaneous selection.
    • Successfully created a homozygous dhfr-ts- line (hyg/neo) auxotrophic for thymidine with high targeting efficiency.

    Conclusions:

    • The double gene targeting method is effective for creating homozygous gene replacements in Leishmania major.
    • This approach facilitates functional genetic testing in various asexual diploids, including fungi and mammalian cells.
    • The method holds potential for developing improved live vaccines against leishmaniasis.