Approximately 100 million confirmed infections and 20,000 deaths are caused by Dengue virus (DENV... more Approximately 100 million confirmed infections and 20,000 deaths are caused by Dengue virus (DENV) outbreaks annually. Global warming and rapid dispersal have resulted in DENV epidemics in formally non-endemic regions. Currently no consistently effective preventive measures for DENV exist, prompting development of transgenic and paratransgenic vector control approaches. Production of transgenic mosquitoes refractory for virus infection and/or transmission is contingent upon defining antiviral genes that have low probability for allowing escape mutations, and are equally effective against multiple serotypes. Previously we demonstrated the effectiveness of an anti-viral group I intron targeting U143 of the DENV genome in mediating trans-splicing and expression of a marker gene with the capsid coding domain. In this report we examine the effectiveness of coupling expression of ΔN Bax to trans-splicing U143 intron activity as a means of suppressing DENV infection of mosquito cells. Targ...
Proceedings of the National Academy of Sciences, 2005
Functional analysis of the Plasmodium falciparum genome is restricted because of the limited abil... more Functional analysis of the Plasmodium falciparum genome is restricted because of the limited ability to genetically manipulate this important human pathogen. We have developed an efficient transposon-mediated insertional mutagenesis method much needed for high-throughput functional genomics of malaria parasites. A drug-selectable marker, human dihydrofolate reductase, added to the lepidopteran transposon piggyBac , transformed parasites by integration into the P. falciparum genome in the presence of a transposase-expressing helper plasmid. Multiple integrations occurred at the expected TTAA target sites throughout the genome of the parasite. We were able to transform P. falciparum with this piggyBac element at high frequencies, in the range of 10 -3 , and obtain stable clones of insertional mutants in a few weeks instead of 6–12 months. Our results show that the piggyBac transposition system can be used as an efficient, random integration tool needed for large-scale, whole-genome mu...
Background Much of the Plasmodium falciparum genome encodes hypothetical proteins with limited ho... more Background Much of the Plasmodium falciparum genome encodes hypothetical proteins with limited homology to other organisms. A lack of robust tools for genetic manipulation of the parasite limits functional analysis of these hypothetical proteins and other aspects of the Plasmodium genome. Transposon mutagenesis has been used widely to identify gene functions in many organisms and would be extremely valuable for functional analysis of the Plasmodium genome. Results In this study, we investigated the lepidopteran transposon, piggyBac, as a molecular genetic tool for functional characterization of the Plasmodium falciparum genome. Through multiple transfections, we generated 177 unique P. falciparum mutant clones with mostly single piggyBac insertions in their genomes. Analysis of piggyBac insertion sites revealed random insertions into the P. falciparum genome, in regards to gene expression in parasite life cycle stages and functional categories. We further explored the possibility of...
Current control efforts for mosquito-borne arboviruses focus on mosquito control involving insect... more Current control efforts for mosquito-borne arboviruses focus on mosquito control involving insecticide applications, which are becoming increasingly ineffective and unsustainable in urban areas. Mosquito population replacement is an alternative arbovirus control concept aiming at replacing virus-competent vector populations with laboratory-engineered incompetent vectors. A prerequisite for this strategy is the design of robust anti-pathogen effectors that can ultimately be genetically driven through a wild-type population. Several anti-pathogen effector concepts have been developed that target the RNA genomes of arboviruses such as dengue virus in a highly sequence-specific manner. Design principles are based on long inverted-repeat RNA triggered RNA interference, catalytic hammerhead ribozymes, and trans-splicing Group I Introns that are able to induce apoptosis in virus-infected cells following splicing with target viral RNA.
We have developed a system for stable germline transformation in the silkworm Bombyx mori L. usin... more We have developed a system for stable germline transformation in the silkworm Bombyx mori L. using piggyBac, a transposon discovered in the lepidopteran Trichoplusia ni. The transformation constructs consist of the piggyBac inverted terminal repeats flanking a fusion of the B. mori cytoplasmic actin gene BmA3 promoter and the green fluorescent protein (GFP). A nonautonomous helper plasmid encodes the piggyBac transposase. The reporter gene construct was coinjected into preblastoderm eggs of two strains of B. mori. Approximately 2% of the individuals in the G1 broods expressed GFP. DNA analyses of GFP-positive G1 silkworms revealed that multiple independent insertions occurred frequently. The transgene was stably transferred to the next generation through normal Mendelian inheritance. The presence of the inverted terminal repeats of piggyBac and the characteristic TTAA sequence at the borders of all the analyzed inserts confirmed that transformation resulted from precise transpositio...
Approximately 100 million confirmed infections and 20,000 deaths are caused by Dengue virus (DENV... more Approximately 100 million confirmed infections and 20,000 deaths are caused by Dengue virus (DENV) outbreaks annually. Global warming and rapid dispersal have resulted in DENV epidemics in formally non-endemic regions. Currently no consistently effective preventive measures for DENV exist, prompting development of transgenic and paratransgenic vector control approaches. Production of transgenic mosquitoes refractory for virus infection and/or transmission is contingent upon defining antiviral genes that have low probability for allowing escape mutations, and are equally effective against multiple serotypes. Previously we demonstrated the effectiveness of an anti-viral group I intron targeting U143 of the DENV genome in mediating trans-splicing and expression of a marker gene with the capsid coding domain. In this report we examine the effectiveness of coupling expression of ΔN Bax to trans-splicing U143 intron activity as a means of suppressing DENV infection of mosquito cells. Targ...
Proceedings of the National Academy of Sciences, 2005
Functional analysis of the Plasmodium falciparum genome is restricted because of the limited abil... more Functional analysis of the Plasmodium falciparum genome is restricted because of the limited ability to genetically manipulate this important human pathogen. We have developed an efficient transposon-mediated insertional mutagenesis method much needed for high-throughput functional genomics of malaria parasites. A drug-selectable marker, human dihydrofolate reductase, added to the lepidopteran transposon piggyBac , transformed parasites by integration into the P. falciparum genome in the presence of a transposase-expressing helper plasmid. Multiple integrations occurred at the expected TTAA target sites throughout the genome of the parasite. We were able to transform P. falciparum with this piggyBac element at high frequencies, in the range of 10 -3 , and obtain stable clones of insertional mutants in a few weeks instead of 6–12 months. Our results show that the piggyBac transposition system can be used as an efficient, random integration tool needed for large-scale, whole-genome mu...
Background Much of the Plasmodium falciparum genome encodes hypothetical proteins with limited ho... more Background Much of the Plasmodium falciparum genome encodes hypothetical proteins with limited homology to other organisms. A lack of robust tools for genetic manipulation of the parasite limits functional analysis of these hypothetical proteins and other aspects of the Plasmodium genome. Transposon mutagenesis has been used widely to identify gene functions in many organisms and would be extremely valuable for functional analysis of the Plasmodium genome. Results In this study, we investigated the lepidopteran transposon, piggyBac, as a molecular genetic tool for functional characterization of the Plasmodium falciparum genome. Through multiple transfections, we generated 177 unique P. falciparum mutant clones with mostly single piggyBac insertions in their genomes. Analysis of piggyBac insertion sites revealed random insertions into the P. falciparum genome, in regards to gene expression in parasite life cycle stages and functional categories. We further explored the possibility of...
Current control efforts for mosquito-borne arboviruses focus on mosquito control involving insect... more Current control efforts for mosquito-borne arboviruses focus on mosquito control involving insecticide applications, which are becoming increasingly ineffective and unsustainable in urban areas. Mosquito population replacement is an alternative arbovirus control concept aiming at replacing virus-competent vector populations with laboratory-engineered incompetent vectors. A prerequisite for this strategy is the design of robust anti-pathogen effectors that can ultimately be genetically driven through a wild-type population. Several anti-pathogen effector concepts have been developed that target the RNA genomes of arboviruses such as dengue virus in a highly sequence-specific manner. Design principles are based on long inverted-repeat RNA triggered RNA interference, catalytic hammerhead ribozymes, and trans-splicing Group I Introns that are able to induce apoptosis in virus-infected cells following splicing with target viral RNA.
We have developed a system for stable germline transformation in the silkworm Bombyx mori L. usin... more We have developed a system for stable germline transformation in the silkworm Bombyx mori L. using piggyBac, a transposon discovered in the lepidopteran Trichoplusia ni. The transformation constructs consist of the piggyBac inverted terminal repeats flanking a fusion of the B. mori cytoplasmic actin gene BmA3 promoter and the green fluorescent protein (GFP). A nonautonomous helper plasmid encodes the piggyBac transposase. The reporter gene construct was coinjected into preblastoderm eggs of two strains of B. mori. Approximately 2% of the individuals in the G1 broods expressed GFP. DNA analyses of GFP-positive G1 silkworms revealed that multiple independent insertions occurred frequently. The transgene was stably transferred to the next generation through normal Mendelian inheritance. The presence of the inverted terminal repeats of piggyBac and the characteristic TTAA sequence at the borders of all the analyzed inserts confirmed that transformation resulted from precise transpositio...
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Papers by Malcolm Fraser