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 âÂÂunfavorableâ 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.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/4205 |
| Date | 30 October 2006 |
| Creators | Gray, Christine Elizabeth |
| Contributors | Coates, Craig J. |
| Publisher | Texas A&M University |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | 4196221 bytes, electronic, application/pdf, born digital |
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