Gray, Christine Elizabeth (2005-08). Promoters, enhancers and insulators for improved mosquito transgenesis. Doctoral Dissertation. Thesis uri icon

abstract

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

publication date

  • August 2005