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AN的基因组中。冈比亚亚目。这些目标中更直接的是开发一种基于一个或多个II类转座子的生殖系转化方法，即通过剪切和粘贴机制移动DNA中间体的转座子。我们将首先探索已经用于转化非果蝇昆虫的4个这样的转座子的质到质转座活性：Hermes，Minos，Mos1 Mariner和PiggyBac。这些转座子在An中显示转位移动性。冈比亚胚胎(这已经在Hermes中被记录在案)将在这样的检测中进行更详细的检查，以努力通过改变助手与供体的质粒比来优化质粒到质粒的转座。同时，我们将进行伽马射线诱变筛选。为了产生两种基因中的一种或两种都发生突变的菌株，这两种基因编码对全色素眼色素合成至关重要的酶：色氨酸加氧酶(在黑腹毛虫中为朱红色)和犬尿氨酸羟基酶(在黑腹毛虫中为朱砂)。由此产生的突变株(S)将成为基于上述最有希望的II类转座子的构建物转化实验的目标。我们将使用已经存在的野生型色氨酸加氧酶或犬尿氨酸羟基酶受体构建，并在其他双翅目(Ae.埃及斑潜蝇(Aegypti)和黑腹斑潜蝇D.Blackogaster)。最后，当一个功能正常的AN。为了开发一种基于第二类转座子的冈比亚转化方法，我们将致力于开发一种Cre/lox靶点特异性重组系统，将大片段DNA(超过20kb)“对接”到AN中。冈比亚亚纲基因组。在后一个目标中，我们将以印第安纳大学的露西和彼得·切巴斯博士的结果为指导，他们目前正在为D.Blackogaster和加州大学欧文分校的Anthony A.James开发这样一种方法，他们正在与Ae进行类似的工作。埃及伊蚊。

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.