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A method for high efficiency YAC lipofection into murine embryonic stem cells

J T Lee1, R Jaenisch

  • 1Whitehead Institute for Biomedical Research, Biology Department, Massachusetts Institute of Technology, Cambridge 02142, USA. jlee@wi.mit.edu

Nucleic Acids Research
|December 15, 1996
PubMed
Summary

This study presents an optimized lipofection protocol for introducing yeast artificial chromosomes (YACs) into mouse embryonic stem (ES) cells. The improved method requires less YAC DNA and yields more intact transgenic clones for research.

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Area of Science:

  • * Molecular Biology
  • * Stem Cell Biology
  • * Genetic Engineering

Background:

  • * Yeast artificial chromosomes (YACs) are essential for studying large genomic regions.
  • * Efficient introduction of YACs into murine embryonic stem (ES) cells is crucial for generating transgenic models.
  • * Current lipofection protocols often require substantial YAC DNA and yield limited intact clones.

Purpose of the Study:

  • * To develop a modified lipofection protocol for enhanced YAC delivery into murine ES cells.
  • * To reduce the amount of YAC DNA needed for successful transfection.
  • * To increase the efficiency and yield of correctly integrated transgenic clones.

Main Methods:

  • * Optimization of DNA:cell ratio during lipofection.
  • * Adjustment of condensing agent concentration.

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  • * Modification of ES cell culture conditions.

    • Main Results:

    • * Reduced YAC DNA requirement to a few micrograms.
    • * Improved recovery of neomycin-resistant ES cell colonies.
    • * Increased yield of clones containing both flanking vector markers and the YAC insert.

    Conclusions:

    • * The modified protocol significantly enhances the efficiency of YAC transfer into murine ES cells.
    • * This optimized method allows for the generation of sufficient intact transgenic clones in a single experiment.
    • * Facilitates downstream biological analysis of large genomic fragments in vivo.