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Related Experiment Videos

Cotransformation and gene targeting in mouse embryonic stem cells.

L H Reid1, E G Shesely, H S Kim

  • 1Department of Pathology, University of North Carolina, Chapel Hill 27599.

Molecular and Cellular Biology
|May 1, 1991
PubMed
Summary

Cotransformation using coelectroporation enhances gene targeting efficiency in mammalian cells. This method enriches for targeted cells, aiding in the isolation of nonselectable gene alterations.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • Gene targeting allows precise DNA modification but isolating modified cells can be challenging.
  • Cotransformation, introducing multiple DNA fragments simultaneously, is explored for improved gene targeting efficiency.

Purpose of the Study:

  • To investigate cotransformation for identifying gene-targeted mammalian cells.
  • To assess the enrichment of targeted cells using a cointroduced selectable marker.

Main Methods:

  • Coelectroporation of separate DNA fragments into mammalian cells.
  • Simultaneous introduction of selectable genes and gene targeting constructs.
  • Screening for co-integration of fragments and gene targeting events.

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Main Results:

  • 75% of cells integrated both randomly integrated fragments.
  • Only 4% of gene-targeted cells showed co-integration of a second fragment.
  • An 80-fold enrichment of hypoxanthine phosphoribosyltransferase (hprt)-targeted cells was achieved using a cointroduced neomycin phosphotransferase (neo) gene.

Conclusions:

  • Cotransformation is a valuable tool for enriching gene-targeted cells, particularly those with nonselectable alterations.
  • This protocol significantly improves the efficiency of isolating specific gene-modified cell populations.