photo: James Gathany
The laboratory of David O'Brochta adapts and develops genetic technologies for use in insects to enable the genetic manipulation of insects for multiple purposes - from functional genomic studies to the creation of insects with genotypes and phenotypes that make them useful and valuable for a variety of applications
Genomics holds great promise for transforming our understanding of insect biology, and advances in genome sequencing technologies have generated whole-genome sequence datasets for both model and non-model insects. These data are accumulating at rates that far surpass our abilities to analyze and interpret them, and we currently lack many of the tools, methods and systems to investigate insect gene function empirically.
In addition to genome sequence data, insect scientists need tools, methods and technologies that enable them to determine the function of various components of insect genomes in relation to insect organismal biology, ecology and evolution. Only major insect model systems such as Drosophila melanogaster and Bombyx mori, have a large complement of advanced functional genomics tools such as germ-line transformation, transposon-based gene-, enhancer-, promoter- and protein-trapping, cell ablation, gene mis-expression, site-specific endonuclease-based gene integration and genome modification, user-designed endonuclease-based gene editing, mutagenesis and integration and double-stranded RNA-based gene-silencing. These technologies need to be available for use in non-model insect systems if insect biologists are to fully capitalize on the on-going genomics revolution.
Enhancer Trapping Technology for Anopheles Mosquitoes
Enhancer-trapping (or, more accurately, ‘enhancer-sensing’) allows enhancers to be detected following insertion of a sensor-containing transposon into a region of the genome under the regulatory control of an enhancer. The enhancer-sensor is the open reading frame of the Gal4 transcription factor under the regulatory control of a minimal promoter that results in little or no Gal4 protein being made in the absence of an enhancer. We have used the weak piggyBac transposase promoter present within the 5’ terminal sequences of the transposon to regulate the expression of Gal4. Transposition of the enhancer-trap element into a region of the genome under the regulatory control of an enhancer results in Gal4 expression in a temporal and spatial pattern determined by the target enhancer (Figure). Because Gal4 is a transcription factor its expression is visualized indirectly through expression of a reporter gene (tdTomato) under the regulatory control of a UAS-containing (Gal4-responsive) promoter.
The Gal4 enhancer-trap system permits the 'trapped' enhancer to be used to drive the expression of any transgene under the regulatory control of a Gal4-responsive promoter.
Application 1: Physiological Genetics. We are currently conducting a large enhancer-trap screen to find Anopheles stephensi hemocyte-specific enhancers that will be used to genetically analyze hematopoesis and hemocyte-function in this mosquito. For more about this project >
Application 2: Malaria Vaccine We are currently using Gal4 'drivers' to express various transgenes in Anopheles stephensi to affect the intensity of Plasmodium falciparum infections as part of an effort to improve production of a malaria vaccine consisting of live attenuated Plasmodium facilparum sporozoites. For more about this project >
An enhancer-trap line permitting circulating blood cells to be visualized in vivo.
Gene-trap technologies for identifying and isolating genes expressed in specific temporal and spacial patterns .
We have developed a number of gene-trap systems which we are now using to screen for genes expressed in adult salivary glands as well as other tissues. We are finding our systems to be very efficient.