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Related Experiment Video

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Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange
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Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange.

Tim Pieters1, Lieven Haenebalcke2, Kenneth Bruneel3

  • 1Department of Biomedical Molecular Biology, Ghent University; Inflammation Research Center, VIB; Center for Medical Genetics, Ghent University Hospital; Cancer Research Institute Ghent (CRIG); Tim.Pieters@irc.vib-ugent.be.

Journal of Visualized Experiments : Jove
|May 19, 2017
PubMed
Summary
This summary is machine-generated.

This study introduces a novel gene engineering method for dissecting protein functions. The approach uses recombination-mediated cassette exchange (RMCE) in mouse embryonic stem cells (mESCs) to analyze protein domains and their roles in cellular processes.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • Studying protein function is crucial for understanding cellular mechanisms.
  • Traditional knockout models do not account for protein functional diversity and posttranslational modifications.
  • Existing mouse embryonic stem cell (mESC) lines with ROSA26 locus docking sites facilitate genetic manipulation.

Purpose of the Study:

  • To develop and validate a structure-function approach for dissecting multidomain protein functionalities.
  • To enable the identification of critical protein domains, regulatory pathways, and disease-associated mutations.
  • To provide a method for semi-high throughput analysis of protein function in a knockout background.

Main Methods:

  • Utilized recombination-mediated cassette exchange (RMCE) in ROSA26 locus-targeted mESCs.
  • Generated RMCE-compatible knockout mESCs by crossing with knockout mice.
  • Introduced a panel of candidate rescue constructs into the R26 locus via RMCE targeting.
  • Validated the approach using p120 catenin (p120ctn) knockout mESCs and endoderm differentiation in embryoid bodies.

Main Results:

  • Achieved high targeting efficiencies (close to 100%) for RMCE-mediated insertion of rescue constructs.
  • Demonstrated the ability to introduce multiple constructs in a semi-high throughput manner.
  • Successfully identified functional domains of p120ctn using endoderm differentiation as a phenotypic readout.

Conclusions:

  • The developed structure-function approach effectively dissects multidomain protein functions.
  • This method allows for the identification of key protein domains and their roles in cellular phenotypes.
  • The approach is valuable for studying protein function, identifying disease mechanisms, and discovering therapeutic targets.