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Recycling selectable markers in mouse embryonic stem cells

A Abuin1, A Bradley

  • 1Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA.

Molecular and Cellular Biology
|April 1, 1996
PubMed
Summary
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This study introduces a novel method for creating marker-free mouse stem cell lines with multiple gene mutations. This technique overcomes limitations of traditional gene targeting, enabling advanced genetic analysis and manipulation.

Area of Science:

  • Molecular Biology
  • Genetics
  • Stem Cell Biology

Background:

  • Gene targeting in mammalian cells typically results in permanent selectable marker integration.
  • Multiple gene targeting events can deplete available markers, hindering further genetic studies.
  • Existing methods face limitations in generating cell lines with numerous targeted mutations.

Purpose of the Study:

  • To develop a method for generating mouse embryonic stem cell lines with multiple targeted mutations.
  • To create marker-free cell lines, facilitating subsequent genetic manipulations and analyses.
  • To overcome the limitation of selectable marker exhaustion in sequential gene targeting.

Main Methods:

  • Combined homologous recombination and CRE-loxP-mediated recombination.

Related Experiment Videos

  • Utilized an excisable cassette containing positive and negative selectable markers.
  • Generated homozygous mutations in Rep-3 and mMsh2 loci sequentially.

    • Main Results:

    • Successfully generated mouse embryonic stem cell lines with up to four targeted mutations.
    • All generated cell lines were devoid of exogenous selectable markers.
    • Demonstrated the ability to perform multiple rounds of targeting and marker excision.

    Conclusions:

    • The developed method enables the creation of versatile, marker-free stem cell lines for complex genetic studies.
    • This approach significantly expands the possibilities for genetic engineering in mammalian cells.
    • Marker-free cell lines are crucial for advanced genetic analysis and manipulation.