Virus-based vectors for gene expression in mammalian cells: Semliki Forest virus

Abstract

Publisher Summary Among alphaviruses the most popular members developed into expression vectors are Semliki Forest virus (SFV), Sindbis virus (SIN) and Venezuelan equine encephalitis virus (VEEV). The most commonly used SFV expression system is based on two plasmid vectors: the expression vector contains the SFV non-structural and the foreign genes and the helper vector harbors the SFV structural genes. In vitro-transcribed RNA molecules from both vectors are co-transfected into BHK-21 cells, where immediate synthesis of non-structural SFV proteins occurs. The third version of SFV vectors is the so called DNA-layered vector, where a cytomegalovirus (CMV) promoter or another eukaryotic RNA polymerase II type promoter is utilized to drive the transcription of self-amplifying SFV replicon vectors. A fourth type of SFV expression system has been developed where both in the expression vector and the helper vector the SP6 RNA polymerase promoter was replaced by an RNA polymerase II-dependent promoter. Co-transfection of SFV DNA expression and helper vectors generated titers of 106 particles/ml, which is approximately 100–1000 lower than using RNA-based SFV vectors. This chapter discusses the use of replication-deficient vectors for gene expression both in vitro and in vivo, the expression studies on topologically different recombinant proteins, the use of various mammalian cell lines, and the scale-up for large-scale protein production.. Descriptions of applying SFV vectors for primary cells in cultures, for injection of organotypic hippocampal slices and in vivo expression, recent developments in using SFV vectors for gene therapy, SFV vectors used for retrovirus particle production and applications of both SFV particles as well as nucleic acid vectors (naked RNA and layered DNA vectors) are described. . the engineering of novel less-cytotoxic, temperature-sensitive and down-regulated SFV vectors as well as potential novel applications are discussed in the chapter.