Flexible manipulation of Omb levels in the endogenous expression region of Drosophila wing by combinational overexpression and suppression strategy

Manipulating an exogenous or endogenous gene of interest at a defined level is critical for a wide variety of experiments.The Gal4/UAS system has been widely used to direct gene expression for studying complex genetic and biological problems in Drosophila melanogaster and other model organisms.Driven by a given tissue-specific Gal4,expressing UAS-transgene or UAS-RNAi(RNA interference)could be used to up-or down-regulate target gene expression,respectively.However,the efficiency of the Gal4/UAS system is roughly predefined by properties of transposon vector constructs and the insertion site in the transgenic stock.Here,we describe a simple way to modulate optomotor blind(omb)expression levels in its endogenous expression region of the wing disc.We co-expressed UAS-omb and UAS-omb-RNAi together under the control of dpp-Gal4 driver which is expressed in the omb expression region of the wing pouch.The repression effect is more sensitive to temperature than that of overexpression.At low temperature,overexpression plays a dominant role but the efficiency is attenuated by UAS-omb-RNAi.In contrast,at high temperature RNAi predominates in gene expression regulation.By this strategy,we could manipulate omb expression levels at a moderate level.It allows us to manipulate omb expression levels in the same tissue between overexpression and repression at different stages by temperature control.     

Characterization of a dorsal‐eye Gal4 Line in Drosophila

In Drosophila, the Gal4‐UAS system is used to drive ectopic gene expression in a tissue‐specific manner. In this system, transgenic flies expressing tissue specific Gal4 are crossed to a line in which the gene to be expressed is under the control of a Gal4‐responsive UAS sequence. The resulting progeny express the gene of interest in the pattern of the particular Gal4 line. Since a given UAS‐transgene can be driven by any Gal4 line, this system is predominantly limited by available Gal4 lines. Here we report the characterization of a novel line, DE‐Gal4, which in the eye is expressed in the dorsal compartment for the majority of development. Furthermore, we use functional tests to show that the DE‐Gal4 line is a useful tool with which to manipulate gene expression in half of the developing eye. genesis 48:3–7, 2010. © 2009 Wiley‐Liss, Inc.     

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 expression is rarely tissue-specific enough to allow for precise correlation of the circuit with behavioral readouts. To overcome this major hurdle, we recently developed the FINGR method to achieve a more restrictive Gal4 expression in the tissue of interest. The FINGR method has three components: 1) the traditional Gal4/UAS system; 2) a set of FLP/FRT-mediated Gal80 converting tools; and 3) enhancer-trap FLP (ET-FLP). Gal4 is used to define the primary neural circuitry of interest. Paring the Gal4 with a UAS-effector, such as UAS-MJD78Q or UAS-Shi^ts, regulates the neuronal activity, which is in turn manifested by alterations in the fly behavior. With an additional UAS-reporter such as UAS-GFP, the neural circuit involved in the specific behavior can be simultaneously mapped for morphological analysis. For Gal4 lines with broad expression, Gal4 expression can be restricted by using two complementary Gal80-converting tools: tub^P>Gal80> (''flip out'') and tub^P>stop>Gal80 (''flip in''). Finally, investigators can turn Gal80 on or off, respectively, with the help of tissue-specific ET-FLP. In the flip-in mode, Gal80 will repress Gal4 expression wherever Gal4 and ET-FLP intersect. In the flip-out mode, Gal80 will relieve Gal4 repression in cells in which Gal4 and FLP overlap. Both approaches enable the restriction of the number of cells in the Gal4-defined circuitry, but in an inverse pattern. The FINGR method is compatible with the vast collection of Gal4 lines in the fly community and highly versatile for traditional clonal analysis and for neural circuit mapping. In this protocol, we demonstrate the mapping of FLP expression patterns in select ET-FLPx2 lines and the effectiveness of the FINGR method in photoreceptor cells. The principle of the FINGR method should also be applicable to other genetic model organisms in which Gal4/UAS, Gal80, and FLP/FRT are used.     

Targeted gene expression in the transgenic Aedes aegypti using the binary Gal4-UAS system

In this study, we report the establishment of the binary Gal4/UAS system for the yellow fever mosquito Aedes aegypti. We utilized the 1.8-kb 5′ upstream region of the vitellogenin gene (Vg) to genetically engineer mosquito lines with the Vg-Gal4 activator and established UAS-EGFP responder transgenic mosquito lines to evaluate the binary Gal4/UAS system. The results show that the Vg-Gal4 driver leads to a high level of tissue-, stage- and sex-specific expression of the EGFP reporter in the fat body of Vg-Gal4/UAS-EGFP hybrids after blood-meal activation. In addition, the applicability of this system to study hormonal regulation of gene expression was demonstrated in in vitro organ culture experiments in which the EGFP reporter was highly activated in isolated fat bodies of previtellogenic Vg-Gal4/UAS-EGFP females incubated in the presence of 20-hydroxyecdysone (20E). Hence, this study has opened the door for further refinement of genetic tools in mosquitoes.     

利用Gal4/ VP16- UAS 和双荧光素酶报告基因 系统检测??- 分泌酶活性

目的：确立基于Gal4／vp16-UAS和双荧光素酶报告基因系统检测γ-分泌酶切割淀粉样前体蛋白活性的方法。方法：将插入上游激活序列（SAS）和萤火虫荧光素酶报告基因的质粒MH100,嵌舍酵母活性转录因子（Gal4）、单纯疱疹病毒蛋白（VP16）和γ-分泌酶切割位点的质粒C99-GVP,以度海肾荧光素酶质粒pRL—CMV,用脂质体转染法转入稳定表达淀粉样前体蛋白C末端的人神经母细胞瘤细胞（SH—SYSY）,用免疫沉淀Western blot分析法检测β-淀粉样蛋白（邶）的生成,利用Gal4／vp16-UAS和双荧光素酶报告基因系统测定荧光素酶报告基因的表达。结果：免疫沉淀Westem blot分析表明A（的生成在γ-分泌酶激活荆神经节苷脂GM1作用下升高并呈剂量依赖性,同时双荧光素酶法检测γ-分泌酶活性也同步升高。在γ-分泌酶抑制荆作用下Aβ的产生呈荆量依赖性的减少,同时γ-分泌酶活性也同步降低。结论：基于Gal4／vp16-UAS和双荧光素酶报告基因系统检测γ-分泌酶活性的方法有效可靠,是一种敏感、定量的检测方法。     

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。     

Mosaic analysis and tumor induction in zebrafish by microsatellite instability-mediated stochastic gene expression

Mosaic analysis, in which two or more populations of cells with differing genotypes are studied in a single animal, is a powerful approach to study developmental mechanisms and gene function in vivo. Over recent years, several genetic methods have been developed to achieve mosaicism in zebrafish, but despite their advances, limitations remain and different approaches and further refinements are warranted. Here, we describe an alternative approach for creating somatic mosaicism in zebrafish that relies on the instability of microsatellite sequences during replication. We placed the coding sequences of various marker proteins downstream of a microsatellite and out-of-frame; in vivo frameshifting into the proper reading frame results in expression of the protein in random individual cells that are surrounded by wild-type cells. We optimized this approach for the binary Gal4-UAS expression system by generating a driver line and effector lines that stochastically express Gal4-VP16 or UAS:H2A-EGFP and self-maintaining UAS:H2A-EGFP-Kaloop, respectively. To demonstrate the utility of this system, we stochastically expressed a constitutively active form of the human oncogene H-RAS and show the occurrence of hyperpigmentation and sporadic tumors within 5 days. Our data demonstrate that inducing somatic mosaicism through microsatellite instability can be a valuable approach for mosaic analysis and tumor induction in Danio rerio.KEY WORDS: Mosaic analysis, Microsatellite instability, Lineage tracing, Tumor induction     

Molecular characterization of SMILE as a novel corepressor of nuclear receptors

SMILE (small heterodimer partner interacting leucine zipper protein) has been identified as a coregulator in ER signaling. In this study, we have examined the effects of SMILE on other NRs (nuclear receptors). SMILE inhibits GR, CAR and HNF4α-mediated transactivation. Knockdown of SMILE gene expression increases the transactivation of the NRs. SMILE interacts with GR, CAR and HNF4α in vitro and in vivo. SMILE and these NRs colocalize in the nucleus. SMILE binds to the ligand-binding domain or AF2 domain of the NRs. Competitions between SMILE and the coactivators GRIP1 or PGC-1α have been demonstrated in vitro and in vivo. Furthermore, an intrinsic repressive activity of SMILE is observed in Gal4-fusion system, and the intrinsic repressive domain is mapped to the C-terminus of SMILE, spanning residues 203–354. Moreover, SMILE interacts with specific HDACs (histone deacetylases) and SMILE-mediated repression is released by HDAC inhibitor trichostatin A, in a NR-specific manner. Finally, ChIP (chromatin immunoprecipitation) assays reveal that SMILE associates with the NRs on the target gene promoters. Adenoviral overexpression of SMILE represses GR-, CAR- and HNF4α-mediated target gene expression. Overall, these results suggest that SMILE functions as a novel corepressor of NRs via competition with coactivators and the recruitment of HDACs.     

Domain specific genetic mosaic system in the Drosophila eye

Genetic mosaic approach is commonly used in the Drosophila eye by completely abolishing or misexpressing a gene within a subset of cells to unravel its role during development. Classical genetic mosaic approach involves random clone generation in all developing fields. Consequently, a large sample size needs to be screened to generate and analyze clones in specific domains of the developing eye. To address domain specific functions of genes during axial patterning, we have developed a system for generating mosaic clones by combining Gal4/UAS and flippase (FLP)/FRT system which will allow generation of loss‐of‐function as well as gain‐of‐function clones on the dorsal and ventral eye margins. We used the bifid‐Gal4 driver to drive expression of UAS‐FLP. This reagent can have multiple applications in (i) studying spatio‐temporal function of a gene during dorso‐ventral (DV) axis specification in the eye, (ii) analyzing genetic epistasis of genes involved in DV patterning, and (iii) conducting genome wide screens in a domain specific manner. genesis 51:68–74, 2013. © 2012 Wiley Periodicals, Inc.     

Cell-penetrating DNA-binding protein as a safe and efficient naked DNA delivery carrier in vitro and in vivo

Non-viral gene delivery is a safe and suitable alternative to viral vector-mediated delivery to overcome the immunogenicity and tumorigenesis associated with viral vectors. Using the novel, human-origin Hph-1 protein transduction domain that can facilitate the transduction of protein into cells, we developed a new strategy to deliver naked DNA in vitro and in vivo. The new DNA delivery system contains Hph-1-GAL4 DNA-binding domain (DBD) fusion protein and enhanced green fluorescent protein (EGFP) reporter plasmid that includes the five repeats of GAL4 upstream activating sequence (UAS). Hph-1-GAL4-DBD protein formed complex with plasmid DNA through the specific interaction between GAL4-DBD and UAS, and delivered into the cells via the Hph-1-PTD. The pEGFP DNA was successfully delivered by the Hph-1-GAL4 system, and the EGFP was effectively expressed in mammalian cells such as HeLa and Jurkat, as well as in Bright Yellow-2 (BY-2) plant cells. When 10 μg of pEGFP DNA was intranasally administered to mice using Hph-1-GAL4 protein, a high level of EGFP expression was detected throughout the lung tissue for 7 days. These results suggest that an Hph-1-PTD-mediated DNA delivery strategy may be an useful non-viral DNA delivery system for gene therapy and DNA vaccines.