Electroporation of RNA into Saccharomyces cerevisiae.

Abstract

The cytoplasmic regulation of gene expression has received increased attention in recent years. In order to assess directly the in vivo impact of regulatory elements on the translational efficiency and stability of an mRNA in higher eukaryotes, a variety of methods that deliver RNA directly to the cytoplasm have been developed to avoid any potential complications associated with transcription, pre-mRNA processing, or nucleocytoplasmic transport. Studies in yeast, however, have been largely limited to DNA-based constructs, either as episomes or as genomically integrated genes. Moreover, certain types of mRNAs can not be generated in vivo, e.g., uncapped messages or poly(A)− mRNAs. In vitro translation lysates have been developed for yeast (, , ). However, lysates derived from higher eukaryotes do not reflect the full cytoplasmic regulation observed in vivo. For studies focusing on posttranscriptional regulation in yeast, an RNA-based delivery system provides an in vivo approach to the analysis of posttranscriptional regulatory mechanisms. Electroporation has been used as a DNA delivery method for a wide range of prokaryotes () and eukaryotes (). Electroporation of intact yeast has already proven useful for the introduction of DNA (), protein (), and small molecules (,). The procedure for electroporation of yeast detailed below was originally described by Everett and Gallie ().