利用GAL4-UAS系统在果蝇中过表达研究人类基因功能

随着人类基因组测序的基本完成 ,大量新基因被发现 ,其中许多只有序列及基因组定位信息。新的焦点是这些新基因的功能研究。模式生物果蝇对此起重要作用。利用转基因果蝇和GAL4 UAS系统初步鉴定功能基因 ,建立了源于 10个不同人类基因的共 5 4个转基因果蝇品系 ,然后用 6种不同的GAL4诱导这些转基因在果蝇中过量表达。其中一个人类基因 ,延伸因子 1alpha 1(EF1α 1)的过表达导致果蝇的背板异常和糙眼表型。该研究表明可在果蝇中利用基因过表达策略初筛人类功能基因 ,这为大规模人类基因的功能研究提供了新的手段     

黑腹果蝇二元表达系统原理及应用

二元表达系统是简单、高效的遗传操作工具,提高了特定基因的重组效率,实现了基因精确时空表达。目前应用于黑腹果蝇的二元表达系统主要有GAL4/UAS、FLP/FRT、Lex A/lex Aop、Q系统以及Cre/lox P、CRISPR/Cas9系统,其中GAL4/UAS和FLP/FRT系统应用最为广泛。二元表达系统的联合成功应用于嵌合体的构建,为细胞谱系分析、细胞间相互作用等研究提供了强有力的工具。综述了果蝇二元表达系统的原理、应用及各系统之间的联合应用,为研究者选择遗传操作工具提供了参考。     

Gal80~(ts)与Gal4组合灵敏控制果蝇中UAS转基因的表达水平

【目的】Gal80~(ts)与Gal4组合驱动UAS转基因表达是黑腹果蝇Drosophila melanogaster研究中常用的转基因过表达遗传学工具,通过温度控制实现对UAS转基因表达的灵活开关。Gal80~(ts)是一种温度敏感型蛋白,低温下(18℃)与Gal4蛋白结合并抑制其转录活力,高温下(29℃)解除对Gal4的抑制,从而允许Gal4结合UAS位点,启动UAS转基因的表达。但是从18~29℃的开关只能强烈过表达UAS转基因,而不能灵活调控转基因的表达水平。本实验系统研究一系列温度下转基因的表达水平,从而实现该体系对转基因的表达水平的灵活控制。【方法】以果蝇翅芽这一常用器官组织为研究模型,以2种Gal4品系(dpp-Gal4和en-Gal4,分别由decapentaplgic和engrailed基因的启动子驱动)分别与tub-Gal80~(ts)(微管蛋白基因tubulin启动子驱动)基因重组后,再分别与UAS-wg(wingless)转基因品系杂交;在一系列温度(18,25,27.5,28,28.5和30℃)下进行子代幼虫培养,通过免疫组化染色揭示并量化分析转基因wg在3龄幼虫翅芽上的表达水平。【结果】18~25℃培养条件下,Gal80~(ts)与Gal4组合系统中的UAS转基因不能表达;30℃时培养,转基因强烈地过表达;在25~30℃区间内,随着温度升高,转基因表达水平逐渐上升。【结论】在25~30℃之间的温度调控可以实现对Gal80~(ts)与Gal4组合系统中的UAS转基因表达水平的调控。本研究结果对调控转基因表达程度有重要价值。     

Hsp22对SCA3/MJD转基因果蝇的神经保护作用研究

为了探讨Hsp22在SCA3/MJD发病机制中的作用．选用GMR-GAL4和elav-GAL4驱动子,利用经典的GAL4-UAS系统,将含有78个CAG重复扩增的ataxin-3蛋白片段(MJDtr-Q78)分别在果蝇眼睛和神经系统选择性表达,构建GMR-GAL4/UAS和elav-GAL4/UAS系统SCA3/MJD转基因果蝇模型, 然后利用遗传学方法和热休克反应使Hsp22在SCA3/ MJD转基因果蝇眼睛和神经系统以不同水平过表达．结果表明,Hsp22过表达显著抑制了MJDtr-Q78蛋白的神经毒性,果蝇眼睛视网膜光感受神经元变性明显缓解,果蝇存活能力也显著提高．Hsp22对SCA3/MJD具有保护作用,增强Hsp22表达对SCA3/MJD可能是一种潜在的治疗方法．     

Henna基因突-变是导致北京紫眼果蝇眼部蝶呤含量减少的重要原因

苯丙氨酸羟化酶被认为参与黑腹果蝇眼部蝶呤代谢,但是始终都缺乏有力证据．Henna是苯丙氨酸羟化酶的编码基因,“北京紫眼”果蝇（Hnbp）是Henna基因的一个隐性突变体,Hn^bp突变形成的原因是Henna基因的第二外显子上具有插入片段．在Hn^bp中,其编码产物的Biopterin—Hydroxyl功能域中增加了15个氨基酸残基．蛋白预测结果显示,插入的残基改变了原有的蛋白结构,这种变化很可能降低了苯丙氨酸羟化酶与四氢生物蝶呤结合的能力．为了恢复突变表型,利用UAS—GAL4转基因体系使野生型Henna基因在Hn^bp的背景下表达．在双拷贝的转基因系GMR—GAL4 UAS—Henna／UAS—Henna;Hn^bp／Hn^bp中,突变表型得到完全恢复：果蝇的眼色由突变体的紫色恢复到野生型的红色,蝶呤含量也从突变体的30％升至98％,与野生型差异不显著（P〉0．05）．上述实验结果充分说明,Henna基因突变是导致Hn^bp突变体眼部喋呤含量降低的重要原因,为苯丙氨酸羟化酶参与果蝇眼部蝶呤代谢提供了体内实验的直接证据．     

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。     

研究报告: 沉默信息调节因子2对SCA3/MJD转基因果蝇的神经保护作用及其与自噬的相关性研究

为探讨沉默信息调节因子2(Sir2)在SCA3/MJD发病机制中的作用．选用GMR-GAL4 和Nrv2-GAL4驱动子,利用经典的GAL4-UAS系统,将含有78 个CAG 重复扩增的ataxin-3 蛋白片段(MJDtr-Q78)分别在果蝇眼睛和运动神经元内选择性表达,构建GMR-GAL4/UAS 和Nrv2-GAL4/UAS 系统SCA3/MJD 转基因果蝇模型,然后分别在抑制和不抑制自噬的情况下,使Sir2在SCA3/MJD 转基因果蝇眼睛和运动神经元内过表达．结果发现,Sir2过表达明显抑制了SCA3/MJD 转基因果蝇眼睛视网膜光感受神经元变性,显著改善了果蝇运动能力,而在自噬被抑制后,Sir2的作用效果明显减弱,表明Sir2对SCA3/MJD 转基因果蝇具有神经保护作用,而这种神经保护作用需要依赖自噬的功能．     

人工转录因子促进外源基因在CHO细胞中的高效表达

以酵母转录因子GAL4中1-147位氨基酸序列为构建人工转录因子的DNA结合结构域,单纯疱疹病毒转录激活子VP16中12肽(DALDDFDLDMLG)的4个串联重复作为人工转录因子的功能结构域,用SV40的核定位序列(NLS)将两部分连接起来,构建了人工转录因子GVP4并将其克隆进入表达载体pcDNA3·1/Hygro( )中。将不同长度的人工转录因子结合序列构建在外源基因表达载体pcDNA3·1( )启动子CMV的上游,分别连接外源基因EGFP和tPA。用表达人工转录因子和外源基因的载体共转染CHO细胞,EGFP和tPA在含不同数量人工转录因子结合位点表达载体pcDNA3·1( )转染的CHO细胞中的表达水平呈现不同程度的提高。其中,以引入10个人工转录因子结合位点的表达载体的效果最明显,EGFP和t-PA的表达效率均提高2~3倍。结果表明,人工转录因子能有效地促进外源基因在哺乳动物细胞中的表达。     

基因taf1在果蝇卵巢生殖干细胞分化中的功能初探

果蝇卵巢生殖干细胞(germline stem cell,GSC)是在体(in vivo)研究成体干细胞的理想模型。为挖掘更多有关成体干细胞分化机制的线索,本研究利用果蝇GAL4/UAS表达系统,对编码核心转录起始因子TFIID外周组分的一个基因taf1(TBP-associated factor 1)在GSC分化中的作用及相关机理做了初步探究,结果显示:通过RNAi技术下调生殖细胞系中taf1的表达导致源于GSC的生殖细胞分化受阻,但BMP信号的活性未受影响。进一步的分子检测显示分化促进因子Bam及分化抑制因子Nanos在生殖细胞系中的表达分布改变,提示taf1调控GSC的分化可能依赖Bam和Nanos分子路径。我们的工作将有助于成体干细胞的命运调控机制研究,具有潜在的临床应用价值。     

核定位信号筛选系统的构建

建立了一酵母克隆系统用于克隆含核定位信号 (NLS)的蛋白质的基因 .用表达转录因子GAL4 DNA结合域 - p53(GAL4- DBD- p53)融合蛋白的质粒转化酵母 HF7c,使 GAL4- DBD- p53可结合于报告基因的启动子但因无转录激活域而不能激活转录 .构建一酵母穿梭载体 ,可表达无NLS的 GAL4转录激活域 -大 T抗原 (GAL4- AD- LT)融合蛋白 .融合蛋白基因的下游插入一多克隆位点 .将 c DNA文库插入多克隆位点后 ,如果 c DNA片段可编码 NLS,则 GAL4- AD- LT分子可进入细胞核 ,并通过 LT与 p53的相互作用而使 GAL4- AD结合于启动子和激活报告基因的转录 .构建了这一克隆系统的各质粒 ,并用绿色荧光蛋白 (GFP)验证了其对核内蛋白和胞浆蛋白的甄别能力 .这一系统将有助于从 c DNA文库中筛选编码带有 NLS的蛋白质的基因     

Inducible control of tissue-specific transgene expression in Xenopus tropicalis transgenic lines

Analysis of gene function in vertebrates is facilitated by gain-of-function studies, such as injection of synthetic mRNA in amphibian embryos. This approach is hampered by lack of spatial and temporal control of expression of the introduced gene product. An additional level of control is obtained by nuclear-transfer-mediated transgenesis, but functional analyses are complicated by variability and background abnormalities in primary transgenic embryos. The GAL4/UAS system permits establishment of stable lines and elimination of nuclear-transfer-associated abnormalities, through generation of separate UAS-'effector' and GAL4 'transactivator' transgenic lines. When the GAL4 DNA-binding domain is combined with a steroid hormone ligand-binding domain, this system allows full temporal regulation of transgene expression by introduction of an exogenous steroid analogue, the progesterone antagonist RU486. We show here that by crossing stable Xenopus tropicalis transgenic lines, one bearing a UAS-enhanced cyan fluorescent protein (ECFP) reporter construct, and the other with a GAL4-progesterone receptor fusion driven by a retina-specific promoter, reporter expression in the resulting embryos can be induced with RU486 in a tissue-specific manner. These results suggest that the inducible binary system, in which the target gene expression can be controlled in a stage- and tissue-specific pattern, should be readily applicable for gene function studies at all stages of development.     

Development of a Gateway-compatible two-component expression vector system for plants

Genetic transformation of plants offers the possibility of functional characterization of individual genes and the improvement of plant traits. Development of novel transformation vectors is essential to improve plant genetic transformation technologies for various applications. Here, we present the development of a Gateway-compatible two-component expression vector system for Agrobacterium-mediated plant transformation. The expression system contains two independent plasmid vector sets, the activator vector and the reporter vector, based on the concept of the GAL4/UAS trans-activation system. The activator vector expresses a modified GAL4 protein (GAL4-VP16) under the control of specific promoter. The GAL4-VP16 protein targets the UAS in the reporter vector and subsequently activates reporter gene expression. Both the activator and reporter vectors contain the Gateway recombination cassette, which can be rapidly and efficiently replaced by any specific promoter and reporter gene of interest, to facilitate gene cloning procedures. The efficiency of the activator–reporter expression system has been assessed using agroinfiltration mediated transient expression assay in Nicotiana benthamiana and stable transgenic expression in Arabidopsis thaliana. The reporter genes were highly expressed with precise tissue-specific and subcellular localization. This Gateway-compatible two-component expression vector system will be a useful tool for advancing plant gene engineering.

     

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.     

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.