Development of a more efficient insect transformation system

拟议研究的长期目标是开发物理和遗传学系统，用于将克隆DNA传递到任何昆虫物种，随后将特定位点整合到基因组中。我们将探索黑腹果蝇胚胎的卵黄膜通过最近开发的程序渗透，以吸收DNA和核苷酸前体的能力。我们还将利用两种主要脱氧核糖核酸酶突变的果蝇胚胎来增加体细胞和生殖细胞中引入DNA的持久性。该研究的第三个目标是将酵母的“翻转重组酶”系统应用于昆虫宿主。首先将果蝇和家蝇的胚胎用重组位点序列（FRT）进行转化。然后，将重组酶基因与可选择的标记基因一起引入含有单个FRT序列的质粒中。标记基因的位点特异性重组将通过基因组Southern和染色体原位杂交分析进行监测。由于这种以果蝇为模型系统开发的更有效和更定向的转化系统应该适用于任何其他昆虫物种，因此将基因随意引入害虫和人类疾病载体将成为可能。这种能力将使生物学家能够设计出控制昆虫的新策略

The long term objective of the proposed research is to develop physical and genetics systems for the delivery of cloned DNA to any insect species with subsequent site specific integration into the genome. We will explore the ability of Drosophila melanogaster embryos whose vitelline membranes have been permeabilized by a recently developed procedure to take up both DNA and nucleotide precursors. We will also utilize Drosophila embryos which are mutant for two predominant deoxyribonucleases to increase the persistence of the introduced DNA in both the somatic and germ cells. The third aim of the research is to adapt the "Flip recombinase" system from yeast for use in insect hosts. Initially Drosophila and Musca domestica (housefly) embryos will be transformed with the recombination site sequence (FRT). Then, the recombinase gene will be introduced along with selectable marker genes in plasmids containing a single FRT sequence. Site specific recombination of the marker genes will be monitored by genomic Southern and chromosomal in situ hybridization analysis. Because such a more efficient and site directed transformation system that is developed for Drosophila as a model system should be applicable to any other insect species, it will be possible to introduce genes at will into insect pests and vectors of human disease. This ability should allow biologists to devise new strategies for insect control.***//