A versatile in vivo system for directed dissection of gene expression patterns

Tissue-specific gene expression using the upstream activating sequence (UAS)–GAL4 binary system has facilitated genetic dissection of many biological processes in Drosophila melanogaster. Refining GAL4 expression patterns or independently manipulating multiple cell populations using additional binary systems are common experimental goals. To simplify these processes, we developed a convertible genetic platform, the integrase swappable in vivo targeting element (InSITE) system. This approach allows GAL4 to be replaced with any other sequence, placing different genetic effectors under the control of the same regulatory elements. Using InSITE, GAL4 can be replaced with LexA or QF, allowing an expression pattern to be repurposed. GAL4 can also be replaced with GAL80 or split-GAL4 hemi-drivers, allowing intersectional approaches to refine expression patterns. The exchanges occur through efficient in vivo manipulations, making it possible to generate many swaps in parallel. This system is modular, allowing future genetic tools to be easily incorporated into the existing framework.     

Targeted gene expression using the GAL4/UAS system in the silkworm Bombyx mori

The silkworm Bombyx mori is one of the most well-studied insects in terms of both genetics and physiology and is recognized as the model lepidopteran insect. To develop an efficient system for analyzing gene function in the silkworm, we investigated the feasibility of using the GAL4/UAS system in conjunction with piggyBac vector-mediated germ-line transformation for targeted gene expression. To drive the GAL4 gene, we used two endogenous promoters that originated from the B. mori actin A3 (BmA3) and fibroin light-chain (FiL) genes and the artificial promoter 3xP3. GFP was used as the reporter. In initial tests of the function of the GAL4/UAS system, we generated transgenic animals that carried the UAS-GFP construct plus either BmA3-GAL4 or 3xP3-GAL4. GFP fluorescence was observed in the tissues of GFP-positive animals, in which both promoters drove GAL4 gene expression. Animals that possessed only the GAL4 gene or UAS-GFP construct did not show GFP fluorescence. In addition, as a further test of the ability of the GAL4/UAS system to drive tissue-specific expression we constructed FiL-GAL4 lines with 3xP3-CFP as the transformation marker. FiL-GAL4 x UAS-GFP crosses showed GFP expression in the posterior silk gland, in which the endogenous FiL gene is normally expressed. These results show that the GAL4/UAS system is applicable to B. mori and emphasize the potential of this system for controlled analyses of B. mori gene function.     

piggyBac-based insertional mutagenesis and enhancer detection as a tool for functional insect genomics

Transposon mutagenesis provides a fundamental tool for functional genomics. Here we present a non-species-specific, combined enhancer detection and binary expression system based on the transposable element piggyBac: For the different components of this insertional mutagenesis system, we used widely applicable transposons and distinguishable broad-range transformation markers, which should enable this system to be operational in nonmodel arthropods. In a pilot screen in Drosophila melanogaster, piggyBac mutator elements on the X chromosome were mobilized in males by a Hermes-based jumpstarter element providing piggyBac transposase activity under control of the alpha1-tubulin promoter. As primary reporters in the piggyBac mutator elements, we employed the heterologous transactivators GAL4delta or tTA. To identify larval and adult enhancer detectors, strains carrying UASp-EYFP or TRE-EYFP as secondary reporter elements were used. Tissue-specific enhancer activities were readily observed in the GAL4delta/UASp-based systems, but only rarely in the tTA/TRE system. Novel autosomal insertions were recovered with an average jumping rate of 80%. Of these novel insertions, 3.8% showed homozygous lethality, which was reversible by piggyBac excision. Insertions were found in both coding and noncoding regions of characterized genes and also in noncharacterized and non-P-targeted CG-number genes. This indicates that piggyBac will greatly facilitate the intended saturation mutagenesis in Drosophila.     

Knockdown of ecdysis-triggering hormone gene with a binary UAS/GAL4 RNA interference system leads to lethal ecdysis deficiency in silkworm

Ecdysis-triggering hormone (ETH) is an integration factor in the ecdysis process of most insects, including Bombyx mori (silkworm). To understand the function of the ETH gene in silkworm, we developed an effective approach to knockdown the expression of ETH in vivo based on RNA interference (RNAi) and a binary UAS/GAL4 expression system that has been successfully used in other insect species. Two kinds of transgenic silkworm were established with this method: the effector strain with the ETH RNAi sequence under the control of UAS and the activator strain with the GAL4 coding sequence under the control of Bombyx mori cytoplasmic actin3. By crossing the two strains, double-positive transgenic silkworm was obtained, and their ETH expression was found to be dramatically lower than that of each single positive transgenic parent. Severe ecdysis deficiency proved lethal to the double-positive transgenic silkworm at the stage of pharate second instar larvae, while the single positive transgenic or wild-type silkworm had normal ecdysis. This UAS/GAL4 RNAi approach provides a way to study the function of endogenous silkworm genes at different development stages.     

GAL4/UAS系统在转基因技术中的应用研究进展

GAL4/UAS系统是一种转基因技术体系,其原理是利用特定的启动子或增强子,以组织特异性的方式激活酵母转录激活子GAL4的表达,GAL4又以同样的方式引起GAL4反应元件(UAS)-靶基因的转录。GAL4/UAS系统的关键点在于:GAL4基因和UAS-靶基因分别存在于两个转基因系中。GAL4转基因系中有转录激活子,但没有靶基因;在UAS-靶基因系中,转录激活子不存在,因而靶基因处于沉默状态,只有将GAL4转基因系与UAS-靶基因系进行杂交,才可能产生表达靶基因的后代。本文综述了GAL4/UAS系统的建立及其研究应用。     

灵活操控果蝇翅芽中wingless基因表达水平的策略

【目的】灵活操控靶基因的表达水平对于研究基因的功能十分重要。Gal4/UAS系统已被广泛应用于调控基因表达,可研究果蝇Drosophila等模式生物复杂的生物学问题。受采用载体的特性及插入位点的影响,Gal4或UAS转基因品系在构建好之后,其调控靶基因的能力基本是确定的。本研究旨在在现有Gal4/UAS系统的基础上,开发一种新的策略,实现在果蝇翅芽中灵活操控wingless(wg)基因的表达水平。【方法】用遗传学手段将黑腹果蝇Drosophila melanogaster品系的UAS-wg和UAS-wg-RNAi转基因重组到同一黑腹果蝇品系中。将该重组黑腹果蝇品系与dpp-Gal4黑腹果蝇品系杂交,同时驱动UAS-wg和UAS-wg-RNAi在果蝇幼虫翅芽中共表达。杂交子代幼虫分别放置在不同的温度(18, 25和30℃)下培养。将幼虫翅芽解剖并进行免疫组化染色,测量染色的荧光强度,分析翅芽中wg的表达水平。【结果】在低温(18℃)下,UAS-wg在基因表达调控中起主要作用,wg表现为超表达,但其超表达的效率可被UAS-wg-RNAi有效地削弱。相反,在高温(30℃)下,UAS-wg-RNAi起主导作用,wg的表达受到抑制。并且通过转换温度,可实现wg在翅芽发育的不同阶段在超表达和抑制之间相互转化,从而灵活地操控wg基因在翅芽中的表达水平。【结论】该方法可以灵活操控果蝇翅芽中wg基因的表达水平,对于调控转基因的表达有重要的意义。