is a freelance medical writer in Mamaroneck, New York.
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The use of microarrays has allowed analysis of the expression of many more genes at one time than would be possible with traditional methods. This has resulted in huge amounts of data, presenting the challenges of analysis, interpretation, and organization. Nevertheless, useful information is being dissected out of the massive accumulation of expression levels, including identification of new genes, pathways, and interactions, and development of interesting applications. This will eventually allow the Human Genome Project and the completion of sequencing of the genomes of organisms across all the phyla to realize their full potential. Practical applications in the field of human disease should include diagnostics and treatments, and potentially the still-elusive personal and preventative therapies.
Field ApplicationsHilary Ranson, Vector Group, Liverpool School of Tropical Medicine, Liverpool, UK, and her group are using microarrays to look at insecticide resistance genes in mosquito vectors of disease. Insect resistance to insecticides threatens vector control programs, particularly those aimed at malaria parasite vectors, because programs rely primarily on residual spraying of dwellings and the use of insecticide-treated bed nets. The group's work has been made possible in part because of the availability of genome sequences of the African malaria parasite vector Anopheles gambiae and the dengue vector Aedes aegypti. The scientists are using custom arrays to look for insecticide resistance, which is usually due either to alterations in the target sites, resulting in decreased insecticide binding, or to increased rates of insecticide detoxification. Huge gene families that are very rapidly evolving with recent duplications are involved, so gene family members tend to have sequence similarities, which can result in cross-hybridization problems, says Ranson. They are looking at 300 candidate genes with probes derived from 3′ untranslated regions of specific genes to create a small, focused array platform or “detoxification chip” that can be used for field work. Unique sequence probes, including cDNA and 70-mer oligonucleotides, were designed using software to avoid cross-hybridization. This allows a smaller scale approach to measure the level of insecticide resistance in local areas of disease transmission. They are looking for natural variations of putative resistance genes that are overexpressed in A. gambiae, and possibly other Anopheles species. Candidate genes include all genes coding for cytochrome P450 mono-oxygenases, glutathione S-transferases, and carboxylesterases. Other candidate resistance genes include those involved in oxidative stress response.
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Currently the group has several ongoing collaborations, primarily with groups in Africa. Ph.D. students from the Liverpool School of Tropical Medicine often collect mosquitoes in the field in Africa, then return to Liverpool to process the samples. The microarrays are used to analyze messenger RNAs (mRNAs), so the group uses RNAlater, an RNA stabilization solution, to store the mosquito samples. There is no need for cold storage, Ranson explains, and the samples can be stored for a month at room temperature before processing. The first step is to compare mRNA from insecticide-susceptible laboratory strains of insects to mRNA from insecticide-resistant insects, both from the laboratory and the field.
“There are no scanners in the African labs right now,” Ranson says. “We are working toward a platform to establish the analysis in countries directly.” She says that a goal is to be able to develop a field diagnostic and to be able to determine the insecticide resistance patterns in small, local mosquito populations. Single nucleotide polymorphisms (SNPs) associated with resistance gene expression could be used for field-based diagnosis. One question the group would like to answer is whether resistance to pyrethroid insecticides, which are widely used on bed nets and is occurring across Africa, is due to one or several different mechanisms, and if the mechanism is location-dependent. Some resistance mechanisms may be common and major, whereas others may be local and may not have spread.
Ranson says the technology they are using can also be used to detect infection with specific organisms in mosquito and other insect populations. They are trying to develop another SNP-based platform as a vector-monitoring tool, for example, to detect the presence in mosquitoes of Plasmodium species, the causative agent of malaria. Ranson notes that they have a Bill and Melinda Gates Foundation grant to develop such a tool. Microarray-based platforms can also be used to identify the insect species present in pooled collections from exit traps used in dwellings and villages. The collection is preserved in isopropanol in tubes and sent to laboratories in Durban, South Africa, for processing. They would eventually like to see a field diagnostic kit for African research laboratories or malaria control units to determine both the insecticide resistance and degree of infection with malaria parasites in local mosquito populations.
