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Related Experiment Video

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RiboTag Immunoprecipitation in the Germ Cells of the Male Mouse
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RiboTag: Ribosomal Tagging Strategy to Analyze Cell-Type-Specific mRNA Expression In Vivo.

Elisenda Sanz1, Jonathan C Bean2, Daniel P Carey2

  • 1Department of Cell Biology, Physiology, and Immunology and Neuroscience Institute, Autonomous University of Barcelona, Barcelona, Spain.

Current Protocols in Neuroscience
|June 20, 2019
PubMed
Summary

Ribosome tagging enables in vivo analysis of gene expression in specific cells. This method uses hemagglutinin A (HA)-tagged ribosomal proteins to isolate and study mRNA translation, simplifying complex cell isolation for researchers.

Keywords:
RNA-Seqgene expressionneuron-specific mRNApolyribosome immunoprecipitationribosome tagging

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Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • Ribosome tagging is a powerful in vivo technique for studying gene expression and mRNA translation.
  • Conventional methods for isolating specific cell types are often time-consuming and difficult.
  • This approach allows for the analysis of mRNA in targeted cell populations without laborious isolation.

Purpose of the Study:

  • To describe a protocol for ribosome tagging using hemagglutinin A (HA)-tagged ribosomal proteins.
  • To facilitate the analysis of gene expression and mRNA translation in specific cell types.
  • To provide guidance on technical procedures and data analysis for ribosome tagging.

Main Methods:

  • Utilizing a mouse line (RiboTag) with a Cre-inducible Rpl22-HA allele.
  • Employing adeno-associated virus (AAV) vectors for stereotaxic injection and Cre-mediated activation of Rpl22-HA.
  • Performing immunoprecipitation of polysomes and associated mRNAs using anti-HA antibodies.
  • Analyzing purified mRNAs via quantitative real-time PCR (qRT-PCR) or RNA sequencing (RNA-Seq).

Main Results:

  • Successful tagging of ribosomes in specific cell types in vivo.
  • Facilitation of polysome and mRNA purification from targeted cells.
  • Enabling quantitative analysis of mRNA populations using established molecular techniques.

Conclusions:

  • Ribosome tagging provides an efficient method for studying gene expression and mRNA translation in challenging cell types.
  • The described protocols offer a robust approach for researchers in molecular and cell biology.
  • This technique enhances the ability to investigate cellular mechanisms at the translational level.