Chapter 2 Expression of Exogenous Proteins in Mammalian Cells with the Semliki Forest Virus Vector

Abstract

Publisher Summary Expression of cloned sequences using carrier vectors is one of the central techniques in cellular and molecular biology. This chapter describes the preparation of recombinant Semliki Forest Viruses (SFVs), their usability in different cell types, and shows examples of expression of exogenous proteins in baby hamster kidney (BHK) cells and in primary cultures of fetal rat hippocampal neurons. The recombinant SFV vector with the inserted sequence encoding an exogenous protein and the helper plasmid are both linearized with Spe I ; runoff transcription is performed using SP6 RNA polymerase. The in vitro transcripts are introduced into BHK cells by electroporation. Transfection of the recombinant vector RNA alone results in transient expression of the exogenous protein, driven by the SFV RNA polymerase encoded in the same RNA molecule. If the vector and the helper RNAs are co-introduced into the cells, the SFV structural proteins encoded by the helper will form virions that package the recombinant vector RNA and can be used for nonreproductive infection of other cells.