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A dispensable Plasmodium locus for stable transgene expression.

ABSTRACT: The ribosomal small subunit locus has been used for transgene expression in the rodent malaria parasites, Plasmodium berghei and Plasmodium yoelii, but this strategy utilizes single crossover integration and is thus prone to reversion by plasmid excision. Targeting of the ribosomal subunit locus may also have a negative effect on oocyst development in the mosquito. In P. berghei, the p230 paralog locus has been used for transgene expression. Here, we show that the P. yoelii S1 locus (sporozoite expressed gene 1) (PY05712) is dispensable and can be used for stable transgene expression throughout the parasite life cycle. P. yoelii s1(-) parasites show no defect in blood stage replication, oocyst formation, sporozoite production, or liver stage development when compared to P. yoelii wildtype parasites. Further, we show that a fluorescent transgene can be stably expressed from this site. This demonstrates that the S1 locus can be utilized for stable expression of heterologous genes in rodent malaria parasites.

SUBMITTER: Jacobs-Lorena VY

PROVIDER: S-EPMC2839042 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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A dispensable Plasmodium locus for stable transgene expression.

Jacobs-Lorena Vanessa Y VY Mikolajczak Sebastian A SA Labaied Mehdi M Vaughan Ashley M AM Kappe Stefan H I SH

Molecular and biochemical parasitology 20100104 1

The ribosomal small subunit locus has been used for transgene expression in the rodent malaria parasites, Plasmodium berghei and Plasmodium yoelii, but this strategy utilizes single crossover integration and is thus prone to reversion by plasmid excision. Targeting of the ribosomal subunit locus may also have a negative effect on oocyst development in the mosquito. In P. berghei, the p230 paralog locus has been used for transgene expression. Here, we show that the P. yoelii S1 locus (sporozoite ...[more]

PMID: 20045029

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Project description:BackgroundMurine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone.MethodsA MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection.ResultsVectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene.ConclusionThese studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency.

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A dispensable Plasmodium locus for stable transgene expression.

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A dispensable Plasmodium locus for stable transgene expression.

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