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BEAM: A combinatorial recombinase toolbox for binary gene expression and mosaic genetic analysis.

Luciano C Greig1, Mollie B Woodworth1, Alexandros Poulopoulos1

  • 1Department of Stem Cell and Regenerative Biology and Center for Brain Science, Harvard University, Cambridge, MA, USA; Harvard Medical School, Boston, MA, USA.

Cell Reports
|August 19, 2024
PubMed
Summary

We developed a novel Binary Expression Aleatory Mosaic (BEAM) system for precise cell comparison. This method uses genetic manipulation and distinct fluorescent markers to reliably identify cellular phenotypes and test developmental hypotheses.

Keywords:
CP: Cell biologyCreFlpgenetic analysismosaicismrecombinase

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Area of Science:

  • Molecular Biology
  • Developmental Biology
  • Genetics

Background:

  • Comparative cell analysis is crucial for understanding biological processes.
  • Existing methods for distinguishing cell populations can lack reliability and precision.
  • The need for improved tools to study cell function and developmental mechanisms is ongoing.

Purpose of the Study:

  • To introduce the Binary Expression Aleatory Mosaic (BEAM) system for generating and comparing two distinct cell populations.
  • To enhance the reliability and precision of comparative cell analysis through signal amplification and delayed reporter expression.
  • To validate the utility of BEAM in studying cellular phenotypes and testing developmental hypotheses, such as the cortical protomap hypothesis.

Main Methods:

  • Utilizing DNA delivery via transfection or viral transduction.
  • Employing nested recombinase activity to create genetically distinct cell populations.
  • Implementing red fluorescent protein (RFP) for control cells and green fluorescent protein (GFP) for experimental cells.
  • Incorporating recombinase-dependent signal amplification and delayed reporter expression.

Main Results:

  • BEAM successfully generated two genetically distinct, non-overlapping cell populations for direct comparison.
  • The system demonstrated sharper delineation of control and experimental cells, improving reliability.
  • BEAM effectively identified temporally or spatially aberrant phenotypes, changes in cell proliferation/death, and controlled for procedural variability.
  • Application to the cortical protomap hypothesis revealed cell-autonomous specification of area identity.

Conclusions:

  • The BEAM system offers a powerful and reliable method for comparative cell analysis.
  • BEAM facilitates the identification of subtle or complex cellular phenotypes.
  • The system provides a robust platform for investigating fundamental biological questions, including developmental patterning.