Promoters, enhancers and insulators for improved mosquito transgenesis

摘要

Low level and variable transgene expression plague efforts to produce andcharacterize transgenic lines in many species. When transformation efficiency is high,productive transgenic lines can be generated with reasonable effort. However, mostefforts to date in mosquitoes have resulted in suboptimal levels of transformation. This,coupled with the large space and intensive labor requirements of mosquito colonymaintenance makes the optimization of transformation in mosquitoes a research priority.This study proposes two strategies for improving transgene expression andtransformation efficiency. The first is to explore exogenous promoter/enhancercombinations to direct expression of either the transgene itself, or the transposaserequired for insertion of the transgene into the genome. An extension of this strategy isto investigate the use of a powerful viral transactivating protein and its cognate enhancerto further increase expression of these targets. The second strategy involves theidentification of an endogenous boundary element for use in insulating transgenes andtheir associated regulatory elements. This would mitigate the inappropriate expressionor silencing of many transgenes inserted into ??????unfavorable?????? genomic environments as a consequence of an inability to specifically target the integration of transposons currentlyused in mosquito transgenesis.The IE1 transactivating protein and its cognate enhancer from a baculovirus wereshown to significantly increase expression of a reporter gene from three differentpromoters in cultured mosquito cells. Other heterologous enhancer/promotercombinations resulted in minimal increases or insignificant changes in expression.Orthologues of the vertebrate insulator-binding factor, CTCF, were cloned andcharacterized in two mosquito species, Aedes aegypti and Anopheles gambiae. Theexpression profile of mosquito CTCF is consistent with its role as a putative insulatorbindingprotein. Preliminary binding site studies reveal a C/G-rich binding siteconsistent with that known in vertebrates and indicate that CTCF may bind widespreadsites within mosquito genomes.