polyadenylation signal) or be removed prior to packaging and not transferred (e.g. Extraneous post-transcriptional processing signals present in the vector sequence will produce incomplete genomic transcripts (e.g. Hence sequences that interfere with nuclear export or express products detrimental to packaging cell function or survival will not yield virus. The retroviral genome must be produced by RNA polymerase II in the nucleus of the packaging cell, edited (capped and polyadenylated) and transported to the cytoplasm for assimilation into a mature virion at the plasma membrane.
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In spite of the accommodating nature of the retroviral genome, there are inherent limitations in the retroviral life cycle that constrain vector design and impede full utilization of the system. Since their inception more than 20 years ago, retrovirus vectors have been developed to transfer various genetic elements for varied purposes: Stable expression of cDNA for gene studies and therapy efficient expression of short hairpin RNA (shRNA) to trigger RNA interference integration of splice donor sequences for gene trapping generation of gene reporter cell lines. Retroviral vector systems are routinely used as delivery vehicles for efficient and stable gene transfer into mammalian cells. 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. For example, novel reporters with alternatively spliced exon-intron configurations could readily be transduced into virtually any cell. The applications of this technique are not limited to producing the higher levels of transgene expression demonstrated here. Infectious retrovirus carrying the same cassette is readily produced when packaging cells are transfected with in vitro transcribed retroviral genomic RNA. Retroviral vectors carrying an optimized high-level expression cassette do not produce infectious virions when introduced into packaging cells by transfection of DNA. 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. Introduction of the in vitro produced uncapped retroviral genomic transcript into the packaging cells did not lead to any detectable GFP expression.
When the conventional retroviral vector was transfected into packaging cells, the expression cassette drove strong GFP expression, but no infectious retrovirus was produced. 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. We produced an expression cassette comprising a strong enhancer/promoter, an optimised intron, the GFP open reading frame and a strong polyadenylation signal. Resultsīy 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.
Furthermore strong enhancer/promoters within the retroviral payload lead to detrimental competition with the retroviral enhancer/promoter. The retroviral RNA genome is produced by cellular transcription and post-transcriptional 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. However, genetic elements required for high-level gene expression are incompatible with standard systems. Retroviruses are widely used to transfer genes to mammalian cells efficiently and stably.
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