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RUSH and CRUSH: a rapid and conditional RNA interference method in mice.

Juliana R Brown1, Bernd Zetsche, Laurie Jackson-Grusby

  • 1Pathology Department and Kirby Center for Neuroscience, Boston Children's Hospital, Boston, Massachusetts; Harvard Stem Cell Institute, Harvard Medical School, Boston, Massachusetts; Pathology Department, Harvard Medical School, Boston, Massachusetts.

Genesis (New York, N.Y. : 2000)
|October 30, 2013
PubMed
Summary

Researchers developed rapid, conditional, and reversible RNA interference (RNAi) methods for gene silencing in mouse models. These RUSH and CRUSH RNAi systems accelerate functional gene studies in development.

Keywords:
ES cellsRNAimouse modelstransgenesis

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Area of Science:

  • Developmental Biology
  • Genetics
  • Molecular Biology

Background:

  • Conventional knockout mouse technology is time-consuming and may yield off-target effects.
  • RNA interference (RNAi) offers a method to phenocopy mutations but requires optimization for specificity and control.
  • Conditional and reversible gene silencing is crucial for studying gene function in complex biological systems.

Purpose of the Study:

  • To develop a rapid method for conditional and reversible gene silencing in RNAi transgenic mouse models and embryonic stem (ES) cells.
  • To create RUSH (reversible) and CRUSH (conditional) RNAi vectors for precise gene knockdown.
  • To validate the utility of these systems for studying gene function in development.

Main Methods:

  • Designed RUSH and CRUSH RNAi vectors for reversible and conditional gene knockdown.
  • Utilized targeted replacement in ROSA26(lacZ) and V6.5 ES cell lines.
  • Validated RUSH by reversible knockdown of Dnmt1 in vitro.
  • Generated CRUSH transgenic founders using ES cell lines from Cre transgenic strains and tetraploid complementation.
  • Assessed knockdown efficiency in CRUSH(GFP) mice using fluorescence and Western blotting.

Main Results:

  • Demonstrated successful targeted replacement of RUSH and CRUSH vectors in ES cells.
  • Validated reversible Dnmt1 knockdown in vitro using RUSH.
  • Expedited conditional mouse model generation using CRUSH and Cre-driven ES cell lines.
  • Showed quantitative knockdown of GFP fluorescence in CRUSH(GFP) mice, confirming system efficacy.
  • Confirmed knockdown by Western blotting.

Conclusions:

  • RUSH and CRUSH RNAi systems provide rapid, conditional, and reversible gene silencing.
  • These methods enable precise control over gene function studies in mouse models and ES cells.
  • The ability to turn alleles on or off with Cre recombinase facilitates rapid investigation of tissue-specificity and cell autonomy in development.