Enhanced gene expression from retroviral vectors
Abstract
Background: Retroviruses are widely used to transfer genes to mammalian cells efficiently and
stably. However, genetic elements required for high-level gene expression are incompatible with
standard systems. The retroviral RNA genome is produced by cellular transcription and posttranscriptional
processing within packaging cells: Introns present in the retroviral genomic
transcript are removed by splicing, while polyadenylation signals lead to the production of
ineffective truncated genomes. Furthermore strong enhancer/promoters within the retroviral
payload lead to detrimental competition with the retroviral enhancer/promoter.
Results: By exploiting a new method of producing the retroviral genome in vitro it is possible to
produce infectious retroviral particles carrying a high-level expression cassette that completely
prohibits production of infectious retroviral particles by conventional methods.
We produced an expression cassette comprising a strong enhancer/promoter, an optimised intron,
the GFP open reading frame and a strong polyadenylation signal. This cassette was cloned into both
a conventional MMLV retroviral vector and a vector designed to allow in vitro transcription of the
retroviral genome by T7 RNA polymerase.
When the conventional retroviral vector was transfected into packaging cells, the expression
cassette drove strong GFP expression, but no infectious retrovirus was produced. Introduction of
the in vitro produced uncapped retroviral genomic transcript into the packaging cells did not lead
to any detectable GFP expression. However, infectious retrovirus was easily recovered, and when
used to infect target primary human cells led to very high GFP expression – up to 3.5 times greater
than conventional retroviral LTR-driven expression.
Conclusion: Retroviral vectors carrying an optimized high-level expression cassette do not
produce infectious virions when introduced into packaging cells by transfection of DNA. Infectious
retrovirus carrying the same cassette is readily produced when packaging cells are transfected with
in vitro transcribed retroviral genomic RNA. The applications of this technique are not limited to
producing the higher levels of transgene expression demonstrated here. For example, novel
reporters with alternatively spliced exon-intron configurations could readily be transduced into
virtually any cell. Furthermore, because the in vitro transcripts are not translated within the
packaging cells, retroviruses carrying genes lethal to the packaging cells can also be produced.
Citation
BMC Biotechnology 2008 8(19)Publisher
BioMed CentralCollections
Copyright 2008 Blø et al; licensee BioMed Central Ltd.