Year: 2006
Type: dissertation
Abstract: Low level and variable transgene expression plague efforts to produce and\ncharacterize transgenic lines in many species. When transformation efficiency is high,\nproductive transgenic lines can be generated with reasonable effort. However, most\nefforts to date in mosquitoes have resulted in suboptimal levels of transformation. This,\ncoupled with the large space and intensive labor requirements of mosquito colony\nmaintenance makes the optimization of transformation in mosquitoes a research priority.\nThis study proposes two strategies for improving transgene expression and\ntransformation efficiency. The first is to explore exogenous promoter/enhancer\ncombinations to direct expression of either the transgene itself, or the transposase\nrequired for insertion of the transgene into the genome. An extension of this strategy is\nto investigate the use of a powerful viral transactivating protein and its cognate enhancer\nto further increase expression of these targets. The second strategy involves the\nidentification of an endogenous boundary element for use in insulating transgenes and\ntheir associated regulatory elements. This would mitigate the inappropriate expression\nor silencing of many transgenes inserted into “unfavorable” genomic environments as a consequence of an inability to specifically target the integration of transposons currently\nused in mosquito transgenesis.\nThe IE1 transactivating protein and its cognate enhancer from a baculovirus were\nshown to significantly increase expression of a reporter gene from three different\npromoters in cultured mosquito cells. Other heterologous enhancer/promoter\ncombinations resulted in minimal increases or insignificant changes in expression.\nOrthologues of the vertebrate insulator-binding factor, CTCF, were cloned and\ncharacterized in two mosquito species, Aedes aegypti and Anopheles gambiae. The\nexpression profile of mosquito CTCF is consistent with its role as a putative insulatorbinding\nprotein. Preliminary binding site studies reveal a C/G-rich binding site\nconsistent with that known in vertebrates and indicate that CTCF may bind widespread\nsites within mosquito genomes.
Author: Christine E Gray
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2287860811