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Related Experiment Videos

Hygromycin B-selected cell lines from GAL4-regulated pUAST constructs.

Panagiota Makridou1, Camilla Burnett, Tim Landy

  • 1MRC Laboratory for Molecular Cell Biology and Department of Physiology, University College London, Gower Street, London WC1E 6BT, UK.

Genesis (New York, N.Y. : 2000)
|June 24, 2003
PubMed
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Researchers developed stable Drosophila S2 cell lines for easy protein expression using the UAS-GAL4 system and hygromycin selection. This method simplifies protein studies by providing readily accessible protein pools, reducing transfection time and cost.

Area of Science:

  • * Molecular Biology
  • * Cell Biology
  • * Drosophila Genetics

Background:

  • * Traditional transfection methods for Drosophila S2 cells are time-consuming and costly.
  • * Establishing stable cell lines with consistent protein expression is challenging.
  • * The UAS-GAL4 system offers inducible gene expression but requires stable integration for constitutive production.

Purpose of the Study:

  • * To generate stable, selectable Drosophila S2 cell lines for constitutive coexpression of multiple proteins.
  • * To establish a streamlined method for producing readily accessible protein pools.
  • * To reduce the complexity and cost associated with protein-based studies in Drosophila S2 cells.

Main Methods:

  • * Utilized the UAS-GAL4 system with the Actin5C-GAL4 driver for ubiquitous protein expression.

Related Experiment Videos

  • * Cloned a hygromycin resistance gene into the pUAST vector for selection.
  • * Cotransfected S2 cells with pUAST constructs and selected using hygromycin.
  • * Maintained cell cultures in hygromycin-supplemented, serum-free media.

    • Main Results:

    • * Successfully generated stable Drosophila S2 cell lines exhibiting coexpression of multiple proteins.
    • * Demonstrated uniform expression of fluorescent markers (GFP and RFP) in cotransfected cells.
    • * Confirmed concurrent expression of MYC3-tagged proteins via Western blot analysis.
    • * Fluorescent-activated cell sorting (FACS) analysis indicated that 80% of the cell population expressed the GFP marker.

    Conclusions:

    • * The developed method enables the creation of stable, selectable S2 cell lines for constitutive protein production.
    • * This technique provides a valuable tool for facilitating protein-based studies by offering readily accessible protein pools.
    • * The approach significantly reduces the time and expense associated with transient transfections.