Ribosome tagging has become a very useful in vivo approach for analyzing gene expression and mRNA translation in specific cell types that are difficult and time consuming to isolate by conventional methods. The approach is based on selectively expressing a hemagglutinin A (HA)-tagged ribosomal protein in a target cell type and then using antibodies against HA to purify the polysomes and associated mRNAs from the target cell. The original approach makes use of a mouse line (RiboTag) harboring a modified allele of Rpl22 (Rpl22-HA) that is induced by the action of Cre recombinase. The Rpl22-HA gene can also be introduced into the animal by stereotaxic injection of an AAV-DIO-Rpl22-HA that is then activated in Cre-expressing cells. Both methods for tagging ribosomes facilitate the immunoprecipitation of ribosome-bound mRNAs and their analysis by qRT-PCR or RNA-Seq. This protocol will discuss the technical procedures and describe important considerations relevant to the analysis of the data. © 2019 by John Wiley & Sons, Inc.
Keywords: RNA-Seq; gene expression; neuron-specific mRNA; polyribosome immunoprecipitation; ribosome tagging.
© 2019 John Wiley & Sons, Inc.