Identification and characterization of germline-specific promoters for remobilization of transgenes in the mosquitoes, Aedes aegypti and Anopheles gambiae



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The development of genetic transformation systems in insects has revolutionized the field of entomology. Transgenic insects provide tools to identify, isolate and analyze insect genes and to genetically modify insects for the purposes of insect control or disease vector modification. When transformation frequencies are high, multiple transgenic lines can be generated with relative ease. However, in most mosquito species, the results of transformation experiments have been suboptimal. Increased mosquito transformation efficiency is a research priority. Additionally, incorporation of refractory transgenes will not be sufficient to modify natural populations. A gene drive system will be required to allow transgenes to proliferate throughout populations and potentially reach fixation. This study proposes the use of germline-specific regulatory elements to promote confined, regulated transposase expression within the germ tissue. Creation of helper constructs utilizing endogenous promoters will potentially increase genetic transformation frequencies. The generation of lab strains of mosquitoes expressing an endogenous source of transposase within the germline will also serve as a powerful research tool. Endogenous sources of transposase will allow for comparative analysis of integration rates using different donor plasmids. Finally, the generation of autonomous transposable elements will provide a gene drive mechanism to move a tightly-linked refractory gene into a population. Four genes have been identified, cloned, and characterized, revealing expression patterns expected of germline-specific genes. Transcription profiles and in situ hybridization data support these conclusions. Putative cis-acting regulatory elements have been cloned and incorporated into DNA plasmid constructs. These elements are cloned in a manner such that they will regulate fluorescent gene expression. Additionally, similar elements have been cloned upstream of the Mos1 open reading frame, within the inverted terminal repeats of the mariner transposable element, thus creating autonomous elements and a potential gene drive mechanism.