[4] Preparation and use of nuclease-treated rabbit reticulocyte lysates for the translation of eukaryotic messenger RNA

Abstract

The nuclease-treated rabbit reticulocyte lysate supplemented with heterologous tRNA is the most widely used translation system for eukaryotic mRNAs. This chapter describes the procedures to make the lysate, how to make it dependent on added mRNA, and provide some suggestions for methods of preparing gel-filtered nuclease-treated lysates. The ideal translation system is a reticulocyte lysate from which the endogenous mRNA is removed by fractionation or selective destruction without impairing the intrinsic high activity of the translation machinery. The resulting nuclease-treated lysate has a very low activity, unless eukaryotic mRNAs are added. The reticulocytes are highly specialized cells, and the translation machinery shows little specialization or preference with regard to initiation of protein synthesis on different eukaryotic mRNAs. It is only with respect to the complement of tRNA species that the reticulocyte lysate shows special properties, which may impair its ability to translate heterologous mRNA.