AN. AGMBIAE GERM LINE TRANSFORMATION

该项目的长期目标是开发两种不同的方法将新的DNA整合到安。冈比亚按蚊。这些目标中更直接的是开发一种基于一个或多个II类转座子的种系转化方法，这些转座子通过剪切-粘贴机制在DNA中间体中移动。我们将首先探索4个这样的转座子的质粒到质粒转座活性，这些转座子已经被用于转化非果蝇昆虫：Hermes， Minos， Mos1 mariner和piggyBac。这些转座子在An中表现出转位移动性。冈比亚胚胎（这已经为Hermes记录）将在这样的实验中进行更详细的检查，以通过改变辅助质粒与供体质粒的比例来优化质粒到质粒的转位。与此同时，我们将对安进行伽马射线诱变筛查。这两种基因编码对普通色素合成至关重要的酶：色氨酸加氧酶（D. melanogaster的朱砂酶）和犬尿氨酸羟化酶（D. melanogaster的朱砂酶）。由此产生的突变株将成为转化实验的目标，其结构基于上述最有希望的II类转座子。我们将使用野生型色氨酸加氧酶或犬尿氨酸羟化酶受体构建，这些受体已经存在并已在其他双翅目（Ae）中得到验证。埃及伊蚊和黑腹伊蚊)。最后，当一个泛函An。基于II类转座子的Gambiae转化方法是可用的，我们将致力于开发Cre/lox靶向位点特异性重组系统，用于将大片段DNA（超过20kb）“对接”到An中。冈比亚按蚊的基因组。在后一个目标中，我们将以博士的结果为指导。印第安纳大学的露西和彼得·切巴斯（Lucy and Peter Cherbas）和加州大学欧文分校（UC Irvine）的安东尼·a·詹姆斯（Anthony a . James）正在研究这种方法。蚊。

The long term objective of this project is to develop two different methods for integrating new DNA into the genome of An. Gambiae. The more immediate of these objectives is to develop a germ line transformation method based on one or more Class II transposons,, transposons that move dia a DNA intermediate through a cut-and-paste mechanism. We will initially explore plasmid-to-plasmid transpositional activity of 4 such transposons that have already been used to transform non-drosophilid insects: Hermes, Minos, Mos1 mariner, and piggyBac. These transposons that show transpositional mobility in An. Gambiae embryos (this has already been documented for Hermes) will be examined in more detail in such assays in an effort to optimize plasmid- to-plasmid transposition by varying helper-to-donor plasmid ratios. At the same time, we will be conducting a gamma-ray mutagenesis screen of An. Gambiae in order to produce strains with a mutations in one or both of two genes that encode enzymes important for ommochrome eye pigment synthesis: tryptophan oxygenase (vermillion in D. melanogaster) and kynurenine hydroxylase (cinnabar in D. melanogaster). The resulting mutant strain(s) will then be the target of transformation experiments with constructs based on the most promising of the above Class II transposons. We will use wild type tryptophan oxygenase or kynurenine hydroxylase receptor constructs that already exit and have been validated in other Diptera (Ae. Aegypti and D. melanogaster). Finally, when a functional An. Gambiae transformation method based on a Class II transposons is available, we will undertake to develop a Cre/lox target site-specific recombination system for "docking" large fragments of DNA (in excess of 20 kb) in to the An. Gambiae genome. In this latter aim, we will be guided by the results of Drs. Lucy and Peter Cherbas at Indiana University who are currently developing such a method for D. melanogaster and Anthony A. James at UC Irvine who is doing similar work with Ae. Aegypti.