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Enhanced gene expression from retroviral vectors.

Blø M, Micklem DR, Lorens JB - BMC Biotechnol. (2008)

Bottom Line: 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.Introduction of the in vitro produced uncapped retroviral genomic transcript into the packaging cells did not lead to any detectable GFP expression.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedicine, University of Bergen, Bergen, Norway. Magnus.Bloe@biomed.uib.no

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 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. 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.

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A) Packaging cell expression. Packaging cells were transfected with equal amounts of pC-GFP, pC-cGreen and pC-neerGc retroviral vector plasmids. GFP expression was quantified in single cells at 24 hours by flow cytometry. B) Expression levels in infected cells. At 18 hours post-transfection, conditioned medium was harvested from the packaging cells and used to infect human umbilical vein cells (HUVEC). GFP expression was quantified in single cells at 48 hours post-infection by flow cytometry. pC-GFP infected cells expresses strong GFP levels, while neither the pC-cGreen nor the pC-neerGc retroviral vector produced detectable viral infection. ND: none detected. C) A high expression retroviral vector using in vitro transcribed genomes. Early passage primary endothelial cells (HUVEC) were infected for 24 hours using serial dilutions of virus generated by the pC-GFP, pT-GFP, pT-cGreen and pT-neerGc vectors. The GFP expression level of individual cells harboring single viral integrations was measured by flow cytometry at 48 hours post-infection. GFP expression in the proviruses driven by the optimized CMV promoter/intron/pA cassette (pT-cGreen, pT-neerGc) is substantially higher than that obtained by LTR-promoter dependent expression (pC-GFP, pT-GFP).
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Figure 3: A) Packaging cell expression. Packaging cells were transfected with equal amounts of pC-GFP, pC-cGreen and pC-neerGc retroviral vector plasmids. GFP expression was quantified in single cells at 24 hours by flow cytometry. B) Expression levels in infected cells. At 18 hours post-transfection, conditioned medium was harvested from the packaging cells and used to infect human umbilical vein cells (HUVEC). GFP expression was quantified in single cells at 48 hours post-infection by flow cytometry. pC-GFP infected cells expresses strong GFP levels, while neither the pC-cGreen nor the pC-neerGc retroviral vector produced detectable viral infection. ND: none detected. C) A high expression retroviral vector using in vitro transcribed genomes. Early passage primary endothelial cells (HUVEC) were infected for 24 hours using serial dilutions of virus generated by the pC-GFP, pT-GFP, pT-cGreen and pT-neerGc vectors. The GFP expression level of individual cells harboring single viral integrations was measured by flow cytometry at 48 hours post-infection. GFP expression in the proviruses driven by the optimized CMV promoter/intron/pA cassette (pT-cGreen, pT-neerGc) is substantially higher than that obtained by LTR-promoter dependent expression (pC-GFP, pT-GFP).

Mentions: When transfected into packaging cells, the standard retroviral vectors (pC) produced high levels of GFP expression as measured by flow cytometry (Figure 3A). However, the presence of the internal CMV-expression cassette completely abrogated production of infectious virus (Figure 3B). The complete loss of infectious virus in both pC-cGreen and pC-neerGc is primarily due to the presence of the strong, bidirectional SV40 polyadenylation site [11]. However, competition between the internal CMV promoter and the 5' LTR CMV promoter is also likely to contribute to the loss of titer [7].


Enhanced gene expression from retroviral vectors.

Blø M, Micklem DR, Lorens JB - BMC Biotechnol. (2008)

A) Packaging cell expression. Packaging cells were transfected with equal amounts of pC-GFP, pC-cGreen and pC-neerGc retroviral vector plasmids. GFP expression was quantified in single cells at 24 hours by flow cytometry. B) Expression levels in infected cells. At 18 hours post-transfection, conditioned medium was harvested from the packaging cells and used to infect human umbilical vein cells (HUVEC). GFP expression was quantified in single cells at 48 hours post-infection by flow cytometry. pC-GFP infected cells expresses strong GFP levels, while neither the pC-cGreen nor the pC-neerGc retroviral vector produced detectable viral infection. ND: none detected. C) A high expression retroviral vector using in vitro transcribed genomes. Early passage primary endothelial cells (HUVEC) were infected for 24 hours using serial dilutions of virus generated by the pC-GFP, pT-GFP, pT-cGreen and pT-neerGc vectors. The GFP expression level of individual cells harboring single viral integrations was measured by flow cytometry at 48 hours post-infection. GFP expression in the proviruses driven by the optimized CMV promoter/intron/pA cassette (pT-cGreen, pT-neerGc) is substantially higher than that obtained by LTR-promoter dependent expression (pC-GFP, pT-GFP).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2266726&req=5

Figure 3: A) Packaging cell expression. Packaging cells were transfected with equal amounts of pC-GFP, pC-cGreen and pC-neerGc retroviral vector plasmids. GFP expression was quantified in single cells at 24 hours by flow cytometry. B) Expression levels in infected cells. At 18 hours post-transfection, conditioned medium was harvested from the packaging cells and used to infect human umbilical vein cells (HUVEC). GFP expression was quantified in single cells at 48 hours post-infection by flow cytometry. pC-GFP infected cells expresses strong GFP levels, while neither the pC-cGreen nor the pC-neerGc retroviral vector produced detectable viral infection. ND: none detected. C) A high expression retroviral vector using in vitro transcribed genomes. Early passage primary endothelial cells (HUVEC) were infected for 24 hours using serial dilutions of virus generated by the pC-GFP, pT-GFP, pT-cGreen and pT-neerGc vectors. The GFP expression level of individual cells harboring single viral integrations was measured by flow cytometry at 48 hours post-infection. GFP expression in the proviruses driven by the optimized CMV promoter/intron/pA cassette (pT-cGreen, pT-neerGc) is substantially higher than that obtained by LTR-promoter dependent expression (pC-GFP, pT-GFP).
Mentions: When transfected into packaging cells, the standard retroviral vectors (pC) produced high levels of GFP expression as measured by flow cytometry (Figure 3A). However, the presence of the internal CMV-expression cassette completely abrogated production of infectious virus (Figure 3B). The complete loss of infectious virus in both pC-cGreen and pC-neerGc is primarily due to the presence of the strong, bidirectional SV40 polyadenylation site [11]. However, competition between the internal CMV promoter and the 5' LTR CMV promoter is also likely to contribute to the loss of titer [7].

Bottom Line: 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.Introduction of the in vitro produced uncapped retroviral genomic transcript into the packaging cells did not lead to any detectable GFP expression.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedicine, University of Bergen, Bergen, Norway. Magnus.Bloe@biomed.uib.no

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 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. 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.

Show MeSH
Related in: MedlinePlus