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DNA整合到An的基因组中。冈比亚这些目标中更直接的是开发一种基于一种或多种II类转座子的生殖系转化方法，所述转座子通过剪切-粘贴机制通过DNA中间体移动。我们将首先探讨质粒到质粒的转座活性的4个这样的转座子，已经被用来转化非果蝇昆虫：爱马仕，米诺斯，Mos 1水手，和piggyBac。这些转座子在An.冈比亚胚胎（这已经在爱马仕中记录）将在此类试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-质粒转座。同时，我们将进行一个γ射线诱变筛选的一个。冈比亚酵母，以产生两个基因中的一个或两个突变的菌株，这两个基因编码对眼色素合成很重要的酶：色氨酸加氧酶（在D。melanogaster）和犬尿氨酸羟化酶（D. melanogaster中的朱砂）。melanogaster）。然后，所得的突变株将成为用基于上述II类转座子中最有希望的构建体进行转化实验的靶标。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体，其已经存在并已在其他双翅目（Ae. Aegypti和D. melanogaster）。最后，当一个功能的一个。基于II类转座子的冈比亚转化方法是可用的，我们将致力于开发Cre/lox靶位点特异性重组系统，用于将大片段DNA（超过20 kb）“对接”到An中。冈比亚基因组。在后一个目标中，我们将以印第安纳州大学的Lucy和Peter Cherbas博士的结果为指导，他们目前正在开发一种用于D. melanogaster和Anthony A.加州大学欧文分校的詹姆斯正在与Ae做类似的工作。埃及人

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.