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Related Experiment Videos

Mutant loxP vectors for selectable marker recycle and conditional knock-outs.

H Arakawa1, D Lodygin, J M Buerstedde

  • 1Heinrich-Pette-Institute, Martinistrasse 52, D-20251 Hamburg, Germany. hiroshi@genetics.hpi-uni-hamburg.de

BMC Biotechnology
|October 10, 2001
PubMed
Summary
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Researchers developed new mutant loxP vectors to enable selectable marker recycling and conditional gene knock-outs. This system facilitates efficient gene function studies, especially for essential genes, in various cell lines.

Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • Targeted gene disruption is crucial for studying gene function.
  • The DT40 chicken B cell line is a model for gene knock-outs but limited by selectable markers.
  • Recycling selectable markers using site-specific recombination (e.g., Cre/loxP) is highly desirable.

Purpose of the Study:

  • To develop a system for selectable marker recycling in gene knock-out studies.
  • To enable conditional gene knock-out and complementation strategies.
  • To facilitate the analysis of essential genes.

Main Methods:

  • Constructed plasmid vectors with selectable marker genes (neoR, puroR, bsr) flanked by mutant loxP sites.
  • Utilized Cre recombinase expression for marker gene excision.

Related Experiment Videos

  • Developed a versatile expression vector for cDNA cloning between mutant loxP sites.
  • Designed knock-out constructs combining floxed markers with cDNA expression cassettes.

    • Main Results:

    • Successfully excised selectable marker genes using Cre/loxP recombination, converting mutant loxP sites to inactive double-mutant loxP.
    • Created a single-step gene knock-out and complementation system.
    • Demonstrated the ability to terminate gene expression via Cre-mediated deletion of cDNA cassettes.
    • The system is effective for analyzing essential genes that cause lethality upon disruption.

    Conclusions:

    • Developed mutant loxP vectors for efficient selectable marker recycling and conditional gene knock-out.
    • The system supports a wide range of cell lines due to the use of the beta-actin promoter.
    • This strategy enhances the study of gene function, particularly for essential genes.