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Related Experiment Videos

RNAi-based conditional gene knockdown in mice using a U6 promoter driven vector.

Vivek Shukla1, Xavier Coumoul, Chu-Xia Deng

  • 1Genetics of Development and Disease Branch, 10/9N105, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.

International Journal of Biological Sciences
|February 17, 2007
PubMed
Summary

Conditional RNA interference (RNAi) in mice enables precise gene function studies. This method uses a Cre-LoxP system for inducible small hairpin RNA (shRNA) expression, allowing tissue-specific gene knockdown and analysis.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Developmental Biology

Background:

  • RNA interference (RNAi) is crucial for gene function research across species.
  • Conditional gene expression systems are vital for studying gene roles in specific tissues or developmental stages.
  • Previous work established a Cre-LoxP-based system for conditional small hairpin RNA (shRNA) expression.

Purpose of the Study:

  • To provide a detailed protocol for a simplified, single-step cloning procedure for vector construction.
  • To enable fast and efficient in vivo, tissue-specific gene function deciphering using conditional RNAi.
  • To demonstrate the utility of the system through targeted knockdown of Fibroblast Growth Factor Receptor 2 (Fgfr2).

Main Methods:

  • Utilized a Cre-LoxP system for conditional activation of a U6 promoter-driven small hairpin RNA (shRNA) expression vector.

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  • Incorporated a ploxPneo cassette within the U6 promoter, rendering it inactive until Cre-mediated excision.
  • Developed a simplified single-step cloning procedure for vector construction.

    • Main Results:

    • Demonstrated successful knockdown of over 95% of Fgfr2 transcripts in mouse germlines, resulting in embryonic lethality.
    • Showed that restricting Fgfr2 knockdown to the limb progress zone in live animals led to malformations in forelimb and hindlimb digits.
    • Validated the efficiency and speed of the developed vector construction and gene knockdown method.

    Conclusions:

    • The described method offers a robust and streamlined approach for conditional gene knockdown in vivo.
    • This technique facilitates precise spatiotemporal control over gene silencing, enabling detailed analysis of gene function.
    • The simplified cloning protocol accelerates the generation of tools for investigating gene roles in a tissue-specific manner.