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Related Experiment Videos

Genetic manipulation of mouse embryonic stem cells by mutant lambda integrase.

Nicole Christ1, Peter Dröge

  • 1Institute of Genetics, University of Cologne, Cologne, Germany.

Genesis (New York, N.Y. : 2000)
|March 14, 2002
PubMed
Summary

Mutant lambda integrases can precisely delete genes from mouse embryonic stem cells. This site-specific recombination system enables efficient genome editing and facilitates the selection of modified cells using fluorescence.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Genomics

Background:

  • Site-specific recombination is crucial for genome engineering.
  • Lambda integrase systems offer precise DNA manipulation capabilities.
  • Resistance marker genes are often used in genetic selection but require removal.

Purpose of the Study:

  • To demonstrate the use of mutant lambda integrases for gene deletion in mammalian cells.
  • To investigate the efficiency of integrative and excisive recombination pathways for removing marker genes.
  • To establish a method for selecting cells that have undergone site-specific recombination.

Main Methods:

  • Utilized mutant lambda integrases for site-specific recombination in mouse embryonic stem cells.
  • Inserted a neomycin resistance gene flanked by recombination sites into the ROSA26 locus.

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  • Employed enhanced green fluorescence protein (EGFP) as a reporter for successful recombination.
  • Confirmed genomic modifications using Southern analysis and DNA sequencing.

    • Main Results:

    • Achieved precise deletion of the neomycin resistance gene via integrative and excisive recombination.
    • Demonstrated successful excision of the marker gene, leading to EGFP expression.
    • Confirmed the occurrence of expected strand exchange reactions at the genomic level.
    • Validated the use of EGFP for sorting cells with successful site-specific recombination.

    Conclusions:

    • The lambda integrase system is effective for site-specific gene deletion in mouse embryonic stem cells.
    • This system provides a tool for efficient genome manipulation and marker gene removal.
    • The integrase system can be combined with other recombinases for advanced genomic protocols.