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Viral paratransgenesis in the malaria vector Anopheles gambiae.

Ren X, Hoiczyk E, Rasgon JL - PLoS Pathog. (2008)

Bottom Line: To demonstrate proof-of-concept of the viral paratransgenesis strategy, we used this system to transduce expression of an exogenous gene (enhanced green fluorescent protein; EGFP) in An. gambiae mosquitoes.Wild-type and EGFP-transducing AgDNV virions were highly infectious to An. gambiae larvae, disseminated to and expressed EGFP in epidemiologically relevant adult tissues such as midgut, fat body and ovaries and were transmitted to subsequent mosquito generations.AgDNV will also be extremely valuable as an effective and easy-to-use laboratory tool for transient gene expression or RNAi in An. gambiae.

View Article: PubMed Central - PubMed

Affiliation: The Johns Hopkins Malaria Research Institute, Baltimore, Maryland, United States of America.

ABSTRACT
Paratransgenesis, the genetic manipulation of insect symbiotic microorganisms, is being considered as a potential method to control vector-borne diseases such as malaria. The feasibility of paratransgenic malaria control has been hampered by the lack of candidate symbiotic microorganisms for the major vector Anopheles gambiae. In other systems, densonucleosis viruses (DNVs) are attractive agents for viral paratransgenesis because they infect important vector insects, can be genetically manipulated and are transmitted to subsequent generations. However, An. gambiae has been shown to be refractory to DNV dissemination. We discovered, cloned and characterized the first known DNV (AgDNV) capable of infection and dissemination in An. gambiae. We developed a flexible AgDNV-based expression vector to express any gene of interest in An. gambiae using a two-plasmid helper-transducer system. To demonstrate proof-of-concept of the viral paratransgenesis strategy, we used this system to transduce expression of an exogenous gene (enhanced green fluorescent protein; EGFP) in An. gambiae mosquitoes. Wild-type and EGFP-transducing AgDNV virions were highly infectious to An. gambiae larvae, disseminated to and expressed EGFP in epidemiologically relevant adult tissues such as midgut, fat body and ovaries and were transmitted to subsequent mosquito generations. These proof-of-principle data suggest that AgDNV could be used as part of a paratransgenic malaria control strategy by transduction of anti-Plasmodium peptides or insect-specific toxins in Anopheles mosquitoes. AgDNV will also be extremely valuable as an effective and easy-to-use laboratory tool for transient gene expression or RNAi in An. gambiae.

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Related in: MedlinePlus

EGFP expression in dissected tissues of adult An. gambiae female.Fluorescence is observable in the midgut, hindgut, ovaries and malpighian tubules. Inset: EGFP-positive inclusion in mature oocyte (arrow).
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ppat-1000135-g007: EGFP expression in dissected tissues of adult An. gambiae female.Fluorescence is observable in the midgut, hindgut, ovaries and malpighian tubules. Inset: EGFP-positive inclusion in mature oocyte (arrow).

Mentions: We purified helper and EGFP-transducer virions from transfected Moss55 cells, exposed first-instar An. gambiae larvae to them and assayed emerged adults for EGFP expression by fluorescence microscopy. EGFP expression was observed in approximately 50% of adults (N>100). We observed similar results when virus from Sua5B cells rather than pBAgα was used as helper. EGFP expression was first observed in the fat body, later disseminating to other tissues such as the eye, midgut, hindgut, malpighian tubules and ovaries (Figures 6 and 7).


Viral paratransgenesis in the malaria vector Anopheles gambiae.

Ren X, Hoiczyk E, Rasgon JL - PLoS Pathog. (2008)

EGFP expression in dissected tissues of adult An. gambiae female.Fluorescence is observable in the midgut, hindgut, ovaries and malpighian tubules. Inset: EGFP-positive inclusion in mature oocyte (arrow).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2500179&req=5

ppat-1000135-g007: EGFP expression in dissected tissues of adult An. gambiae female.Fluorescence is observable in the midgut, hindgut, ovaries and malpighian tubules. Inset: EGFP-positive inclusion in mature oocyte (arrow).
Mentions: We purified helper and EGFP-transducer virions from transfected Moss55 cells, exposed first-instar An. gambiae larvae to them and assayed emerged adults for EGFP expression by fluorescence microscopy. EGFP expression was observed in approximately 50% of adults (N>100). We observed similar results when virus from Sua5B cells rather than pBAgα was used as helper. EGFP expression was first observed in the fat body, later disseminating to other tissues such as the eye, midgut, hindgut, malpighian tubules and ovaries (Figures 6 and 7).

Bottom Line: To demonstrate proof-of-concept of the viral paratransgenesis strategy, we used this system to transduce expression of an exogenous gene (enhanced green fluorescent protein; EGFP) in An. gambiae mosquitoes.Wild-type and EGFP-transducing AgDNV virions were highly infectious to An. gambiae larvae, disseminated to and expressed EGFP in epidemiologically relevant adult tissues such as midgut, fat body and ovaries and were transmitted to subsequent mosquito generations.AgDNV will also be extremely valuable as an effective and easy-to-use laboratory tool for transient gene expression or RNAi in An. gambiae.

View Article: PubMed Central - PubMed

Affiliation: The Johns Hopkins Malaria Research Institute, Baltimore, Maryland, United States of America.

ABSTRACT
Paratransgenesis, the genetic manipulation of insect symbiotic microorganisms, is being considered as a potential method to control vector-borne diseases such as malaria. The feasibility of paratransgenic malaria control has been hampered by the lack of candidate symbiotic microorganisms for the major vector Anopheles gambiae. In other systems, densonucleosis viruses (DNVs) are attractive agents for viral paratransgenesis because they infect important vector insects, can be genetically manipulated and are transmitted to subsequent generations. However, An. gambiae has been shown to be refractory to DNV dissemination. We discovered, cloned and characterized the first known DNV (AgDNV) capable of infection and dissemination in An. gambiae. We developed a flexible AgDNV-based expression vector to express any gene of interest in An. gambiae using a two-plasmid helper-transducer system. To demonstrate proof-of-concept of the viral paratransgenesis strategy, we used this system to transduce expression of an exogenous gene (enhanced green fluorescent protein; EGFP) in An. gambiae mosquitoes. Wild-type and EGFP-transducing AgDNV virions were highly infectious to An. gambiae larvae, disseminated to and expressed EGFP in epidemiologically relevant adult tissues such as midgut, fat body and ovaries and were transmitted to subsequent mosquito generations. These proof-of-principle data suggest that AgDNV could be used as part of a paratransgenic malaria control strategy by transduction of anti-Plasmodium peptides or insect-specific toxins in Anopheles mosquitoes. AgDNV will also be extremely valuable as an effective and easy-to-use laboratory tool for transient gene expression or RNAi in An. gambiae.

Show MeSH
Related in: MedlinePlus