Biomedical subjects

David A O'Brochta

Publications and source records attributed to David A O'Brochta.

10 recordsLinked to original sources

PubMed2006

hAT element population genetics in Anopheles gambiae s.l. in Mozambique.

Herves is a functional Class II transposable element in Anopheles gambiae belonging to the hAT superfamily of elements. Class II transposable elements are used as gene vectors in this species and are also being considered as genetic drive agents for spreading desirable genes through natural populations as part of an effort to control malaria transmission. In this study, Herves was investigated in populations of Anopheles gambiae s.s., Anopheles arabiensis and Anopheles merus in Mozambique over a period of 2 years. The copy number of Herves within these three species was approximately 5 copies per diploid genome and did not differ among species or between years. Based on the insertion-site occupancy-frequency distribution and existing models of transposable element dynamics, Herves appears to be transpositionally active currently or, at least recently, in all species tested. Ninety-five percent of the individuals within the populations of the three species tested contained intact elements with complete Herves transposase genes and this is consistent with the idea that these elements are currently active.

Animals↗

PubMed2005

The Drosophila melanogaster cinnabar gene is a cell autonomous genetic marker in Aedes aegypti (Diptera: Culicidae).

The cinnabar gene of Drosophila melanogaster (Meigen) encodes for kynurenine hydroxylase, an enzyme involved in ommochrome biosynthesis. This gene is commonly included as a visible genetic marker in gene vectors used to create transgenic Aedes aegypti (L.) that are homozygous for the khw allele, the mosquito homolog of cinnabar. Unexpectedly, the phenotype of cells expressing kynurenine hydroxylase in transgenic Ae. aegypti is cell autonomous as demonstrated by the recovery of insects heterozygous for the kynurenine hydroxylase transgene with mosaic eye color patterns. In addition, a transgenic gynandromorph was recovered in which one-half of the insect was expressing the kynurenine hydroxylase transgene, including one eye with red pigmentation, whereas the other half of the insect was homozygous khw and included a white eye. The cell autonomous behavior of cinnabar in transgenic Ae. aegypti is unexpected and increases the utility of this genetic marker.

Aedes↗

PubMed2004

An active transposable element, Herves, from the African malaria mosquito Anopheles gambiae.

Transposable elements have proven to be invaluable tools for genetically manipulating a wide variety of plants, animals, and microbes. Some have suggested that they could be used to spread desirable genes, such as refractoriness to Plasmodium infection, through target populations of Anopheles gambiae, thereby disabling the mosquito's ability to transmit malaria. To achieve this, a transposon must remain mobile and intact after the initial introduction into the genome. Endogenous, active class II transposable elements from An. gambiae have not been exploited as gene vectors/drivers because none have been isolated. We report the discovery of an active class II transposable element, Herves, from the mosquito An. gambiae. Herves is a member of a distinct subfamily of hAT elements that includes the hopper-we element from Bactrocera dorsalis and B. cucurbitae. Herves was transpositionally active in mobility assays performed in Drosophila melanogaster S2 cells and developing embryos and was used as a germ-line transformation vector in D. melanogaster. Herves displays an altered target-site preference from the distantly related hAT elements, Hermes and hobo. Herves is also present in An. arabiensis and An. merus with copy numbers similar to that found in An. gambiae. Preliminary data from an East African population are consistent with the element being transpositionally active in mosquitoes.

Africa↗

PubMed2004

Assessing fitness costs for transgenic Aedes aegypti expressing the GFP marker and transposase genes.

The development of transgenic mosquitoes that are refractory to the transmission of human diseases such as malaria, dengue, and yellow fever has received much interest due to the ability to transform a number of vector mosquito species with transposable elements. Transgenic strains of mosquitoes have been generated with molecular techniques that exhibit a reduced capacity to transmit pathogens. These advancements have led to questions regarding the fitness of transgenic mosquitoes and the ability of transformed mosquitoes to compete and effectively spread beneficial genes through nontransformed field populations, the core requirement of a genetically based control strategy aimed at reducing the spread of mosquito-borne human disease. Here we examine the impact of transgenesis on the fitness of Aedes aegypti, a mosquito that transmits yellow fever. Mosquitoes were altered with two types of transgene, the enhanced GFP gene and two transposase genes from the Hermes and MOS1 transposable elements. We examined the effects of these elements on the survivorship, longevity, fecundity, sex ratio, and sterility of transformed mosquitoes and compared results to the nontransformed laboratory strain. We show that demographic parameters are significantly diminished in transgenic mosquitoes relative to the untransformed laboratory strain. Reduced fitness in transgenic mosquitoes has important implications for the development and utilization of this technology for control programs based on manipulative molecular modification.

Aedes↗

PubMed2004

Tn5 as an insect gene vector.

The purpose of this study was to explore alternatives to insect-derived transposable elements as insect gene vectors with the intention of improving existing insect transgenesis methods. The mobility properties of the bacterial transposon, Tn5, were tested in mosquitoes using a transient transposable element mobility assay and by attempting to create transgenic insects. Tn5 synaptic complexes were assembled in vitro in the absence of Mg(2+) and co-injected with a target plasmid into developing yellow fever mosquito, Aedes aegypti, embryos. Target plasmids recovered from embryos a day later were screened for the presence of Tn5. Recombinants (transposition events) were found at a frequency of 1.2 x 10(-3). Some transposition events did not appear to be associated with canonical 9 bp direct duplications at the site of insertion and also were associated with either deletions or rearrangements. A Tn5 element containing the brain-specific transgene, 3 x P3DsRed, was assembled into synaptic complexes in vitro and injected into pre-blastoderm embryos of Ae. aegypti. Of the approximately 900 embryos surviving injection and developing into adults, two produced transgenic progeny. Both transgenic events involved the co-integrations of approximately five elements resulting in nested and tandem arrayed Tn5::3 x P3DsRed elements. This study extends the known host range of Tn5 to insects and makes available to insect biologists and others another eukaryotic genome-manipulation tool. The hyperactivity of synaptic complexes may be responsible for the unusual clustering of elements and managing this aspect of the element's behavior will be important in future applications of this technology to insects.

Aedes↗

PubMed2004

Transformation systems in insects.

Genetic transformation is an important technology that provides unique opportunities to find, isolate, and analyze genes, as well as to create organisms with unique functional characteristics. Insect biologists have been developing genetic transformation technologies that rely extensively on transposable elements. A number of class II transposable elements isolated originally from insects have been converted into broad host range insect gene vectors. Class II transposable elements are particularly amenable to gene vector development, although they suffer from some limitations such as low rates of recombination. Use of these gene vectors requires the physical introduction of the vectors into developing insect embryos by microinjection. Microinjection methods vary to accommodate the unique physical and developmental characteristics of the target insects. All methods rely on the use of fine glass needles in conjunction with micromanipulators and a microscope. A serious constraint on the use of existing systems can be the inefficiency of successfully delivering the gene vectors to the germ cells of the developing embryo. The general method for vector delivery to insect germ cells is described, as well as variations that are useful under some conditions.

Animals↗

PubMed2004

Ectopic expression of a cecropin transgene in the human malaria vector mosquito Anopheles gambiae (Diptera: Culicidae): effects on susceptibility to Plasmodium.

Genetically altering the disease vector status of insects using recombinant DNA technologies is being considered as an alternative to eradication efforts. Manipulating the endogenous immune response of mosquitoes such as the temporal and special expression of antimicrobial peptides like cecropin may result in a refractory phenotype. Using transgenic technology a unique pattern of expression of cecropin A (cecA) in Anopheles gambiae was created such that cecA was expressed beginning 24 h after a blood meal in the posterior midgut. Two independent lines of transgenic An. gambiae were created using a piggyBac gene vector containing the An. gambiae cecA cDNA under the regulatory control of the Aedes aegypti carboxypeptidase promoter. Infection with Plasmodium berghei resulted in a 60% reduction in the number of oocysts in transgenic mosquitoes compared with nontransgenic mosquitoes. Manipulating the innate immune system of mosquitoes can negatively affect their capacity to serve as hosts for the development of disease-causing microbes.

Animals↗

PubMed2003

Post-integration behavior of a Mos1 mariner gene vector in Aedes aegypti.

The post-integration behavior of insect gene vectors will determine the types of applications for which they can be used. Transposon mutagenesis, enhancer trapping, and the use of transposable elements as genetic drive systems in insects requires transposable elements with high rates of remobilization in the presence of transposase. We investigated the post-integration behavior of the Mos1 mariner element in transgenic Aedes aegypti by examining both germ-line and somatic transpositions of a non-autonomous element in the presence of Mos1 transposase. Somatic transpositions were occasionally detected while germ-line transposition was only rarely observed. Only a single germ-line transposition event was recovered after screening 14,000 progeny. The observed patterns of transposition suggest that Mos1 movement takes place between the S phase and anaphase. The data reported here indicate that Mos1 will be a useful vector in Ae. aegypti for applications requiring a very high degree of vector stability but will have limited use in the construction of genetic drive, enhancer trap, or transposon tagging systems in this species.

Aedes↗

PubMed2003

Gene vector and transposable element behavior in mosquitoes.

The development of efficient germ-line transformation technologies for mosquitoes has increased the ability of entomologists to find, isolate and analyze genes. The utility of the currently available systems will be determined by a number of factors including the behavior of the gene vectors during the initial integration event and their behavior after chromosomal integration. Post-integration behavior will determine whether the transposable elements being employed currently as primary gene vectors will be useful as gene-tagging and enhancer-trapping agents. The post-integration behavior of existing insect vectors has not been extensively examined. Mos1 is useful as a primary germ-line transformation vector in insects but is inefficiently remobilized in Drosophila melanogaster and Aedes aegypti. Hermes transforms D. melanogaster efficiently and can be remobilized in this species. This element is also useful for creating transgenic A. aegypti, but its mode of integration in mosquitoes results in the insertion of flanking plasmid DNA. Hermes can be remobilized in the soma of A. aegypti and transposes using a common cut-and-paste mechanism; however, the element does not remobilize in the germ line. piggyBac can be used to create transgenic mosquitoes and occasionally integrates using a mechanism other than a simple cut-and-paste mechanism. Preliminary data suggest that remobilization is infrequent. Minos also functions in mosquitoes and, like the other gene vectors, appears to remobilize inefficiently following integration. These results have implications for future gene vector development efforts and applications.

Animals↗

PubMed2002

The genome sequence of the malaria mosquito Anopheles gambiae.

Anopheles gambiae is the principal vector of malaria, a disease that afflicts more than 500 million people and causes more than 1 million deaths each year. Tenfold shotgun sequence coverage was obtained from the PEST strain of A. gambiae and assembled into scaffolds that span 278 million base pairs. A total of 91% of the genome was organized in 303 scaffolds; the largest scaffold was 23.1 million base pairs. There was substantial genetic variation within this strain, and the apparent existence of two haplotypes of approximately equal frequency ("dual haplotypes") in a substantial fraction of the genome likely reflects the outbred nature of the PEST strain. The sequence produced a conservative inference of more than 400,000 single-nucleotide polymorphisms that showed a markedly bimodal density distribution. Analysis of the genome sequence revealed strong evidence for about 14,000 protein-encoding transcripts. Prominent expansions in specific families of proteins likely involved in cell adhesion and immunity were noted. An expressed sequence tag analysis of genes regulated by blood feeding provided insights into the physiological adaptations of a hematophagous insect.

Animals↗