Post-graduate: Derek Collinge - Gene silencing technologies to control Helicoverpa armigera
Abstract
Stable transformation is an essential tool for molecular biologists workingon non-model organsims. The ability to introduce and express genes of choice in an organism provides means to investigate important molecular questions such as gene function, biochemcial pathway analysis, reporter gene studies and developmental process. my PhD studies have focussed on the transformation of the pest Helcoverpa armigera with the reporter gene EGFP(enhanced green fluorescent protein). There are essentially two parts to the transformation: 1) DNA delivery and 2)targetr gene integration. Biolistics is a technique for DNA delivery that involves coating microscopic gold particles with the DNA of choice and accelerating them at high velocity into the cells. Biolistics has been widely used to transform many kinds of plant tissue, and has had mixed success transforming Drosophilia embryos. Extensive attempts to adapt biolistics to transform H.armigera embryos proved fruitless, with too many technical hurdles to overcome. these difficulties led me to use microinjection delivery of DNA to embryos. Compared to biolistics, microinjection is a lower-throughput technique delivering DNA to individual embryos, however this method is well established, with none of the hurdles raised by biolistics. Results for the microinjection were encouraging, with a high frequency of transient EGFP expression and the generation of two putative EGFP stably transformed H.armigera lines. Following DNA delivery, integration of target genes into insect genomes is commonly mediated by transposon-based gene movement. i used the class II transposan piggyBac to facilitate the movement of the ECFP reporter gene into the genomes of H.armigera embryos aas a visual proof of integration.
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- 2007 Final Reports
CRDC Final Reports submitted in 2007