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Sequential and counter-selectable cassettes for fission yeast.

Hanna Amelina1, Vera Moiseeva1, Laura Catharine Collopy1

  • 1Chromosome Maintenance Group, UCL Cancer Institute, University College London, Paul O'Gorman Building, Huntley Street, London, WC1E 6DD, UK.

BMC Biotechnology
|November 10, 2016
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Summary

Researchers developed new antibiotic resistance cassettes for fission yeast genetics. These novel cassettes prevent recombination with existing ones, enabling sequential gene targeting and simplifying complex strain construction for genetic studies.

Keywords:
DNA replicationFUdRGene disruption and insertionHA, Flag, PK taggingPoint mutationSchizosaccharomyces pombeThymidine kinaseZeocin

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Area of Science:

  • Molecular Biology
  • Genetics
  • Yeast Biology

Background:

  • Fission yeast is a key model organism in genetic research.
  • Antibiotic resistance cassettes are crucial for selecting desired genotypes in yeast.
  • Existing cassettes share promoter/terminator sequences, hindering sequential genetic modifications due to unwanted recombination.

Purpose of the Study:

  • To develop novel selection cassettes for fission yeast that avoid recombination with existing ones.
  • To enable efficient sequential gene targeting in fission yeast.
  • To create tools for simplifying the construction of complex fission yeast strains.

Main Methods:

  • Modified existing antibiotic resistance cassettes by replacing tef promoter and terminator sequences.
  • Generated plasmids for C-terminal gene tagging.
  • Utilized the anti-selection gene HSV-TK for counter-selection with 5-Fluoro-2'-deoxyuridine (FdU).
  • Introduced the Cre-loxP system for marker removal.
  • Developed a two-step transformation method for inducing point mutations.

Main Results:

  • New selection cassettes showed no recombination with tef-containing cassettes, facilitating sequential gene targeting.
  • Combined antibiotic resistance with HSV-TK for dual selection and counter-selection.
  • FdU counter-selection efficiency was enhanced by hydroxyurea treatment.
  • Demonstrated efficient induction of point mutations using negative selectable markers.

Conclusions:

  • The developed plasmid constructs and techniques are essential for sequential gene targeting in fission yeast.
  • These tools significantly simplify the construction of complex fission yeast strains for research.