Creation of a GAL4/UAS-coupled inducible gene expression system for use in Drosophila cultured cell lines
Creation of a GAL4/UAS-coupled inducible gene expression system for use in Drosophila cultured cell lines
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DOI：
10.1002/gene.10148
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发表时间：
2002-09-01
期刊：
影响因子：
1.5
通讯作者：
Duffy, JB
中科院分区：
文献类型：
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作者：
Klueg, KM;Alvarado, D;Duffy, JB
GAL 4/UAS系统作为果蝇靶向基因表达工具的有用性已得到广泛认可（Duffy，2002，本期）。通过将组织特异性GAL 4驱动与稳定整合的基于pUAST的应答构建体相结合，GAL 4/UAS系统（Brand和Perrimon，1993）将基因错误表达转化为用于功能分析的甚至更精确和更强大的工具。在过去的30年里，果蝇作为模式生物的多功能性也通过从各种野生型和突变型果蝇品系以及这些品系内的各种组织开发培养细胞系而得到扩展（Cherbas和Cherbas，1998）。传统上，果蝇培养细胞系中的调节基因表达是通过将感兴趣的编码序列置于组成型启动子的直接控制下来实现的，例如来自肌动蛋白5C基因的pAct 5C（例如，Krasnow等人，1989），或诱导型启动子，如来自金属硫蛋白（mt）基因的pMT（Bunch等，1988年，创建“直接驱动”系统。因为内源性mt基因表达是果蝇对重金属毒性的系统反应的结果（Maroni et al.，1987），可通过将重金属（通常为硫酸铜形式的铜离子（Cu））引入瞬时或稳定转染的细胞系中来实现诱导型mt基因表达（Bunch等，1988年）。使GAL 4/UAS系统适应用于果蝇培养的细胞系的第一种方法涉及在GAL 4响应性UAS盒的控制下编码特定目的蛋白的构建体与组成型GAL 4驱动子的共转染（例如，在犰狳启动子的控制下，我们未发表的结果，或act 5C启动子，约翰逊等人，2000年），以创建一个更灵活的“间接驱动”系统。我们已经将该系统向前推进了另一步，通过将GAL 4表达置于广泛使用的载体pRMHa-3中的mt启动子的控制下来引入诱导子（Bunch等人，1988;“pMT”，下文中）。通过使用这种pMT：GAL 4构建体和编码果蝇Notch组分的构建体（综述于Baron et al.，2002）和EGF受体（dEGFR 1，综述于Klambt，2000）信号转导途径，我们证明了GAL 4/UAS系统可用于在果蝇细胞系中设计诱导型蛋白表达。这种方法也受益于不断增长的pUAST应答构建体库，可用于果蝇中调控基因表达的工程化。在培养的细胞中使用诱导型GAL 4/UAS系统将提供对表达水平和时间的控制，允许当组成型表达时可能引起毒性的蛋白质的较低表达。我们使用编码野生型Delta蛋白的表达构建体（DeltaWT; Kopczynski et al.，1988）和Notch蛋白的组成型活性形式（NotchICD; Fortini等，1993）来比较我们开发的“间接驱动”pMT：GAL 4/pUAST系统与“直接驱动”pMT表达系统的功效（Bunch等，1988年）。pMT：Δ WT和pMT：NotchICD构建体已广泛用于果蝇培养的细胞中以研究Delta-Notch相互作用和下游信号传导事件（Fehon等人，1990年; Koburg等人，1998; Koburg和Muskavitch，1999; Lieber等人，2002年）。pMT：Δ WT或pMT：GAL 4和PUAST：将DeltaWT构建体转染到果蝇S3细胞中，连续诱导，并通过Western印迹分析随时间测定蛋白质表达水平（图1）。我们发现，当使用等摩尔量的Delta编码构建体时，由pMT：DeltaWT驱动的表达水平...
The usefulness of the GAL4/UAS system as a tool for targeted gene expression in Drosophila melanogaster has been widely recognized (Duffy, 2002, this issue). By combining tissue-specific GAL4 drivers with stably integrated pUAST-based responder constructs, the GAL4/UAS system (Brand and Perrimon, 1993) transformed gene misexpression into an even more precise and powerful tool for functional analysis. Over the past 30 years, the versatility of Drosophila as a model organism has been extended as well by the development of cultured cell lines from a variety wildtype and mutant fly strains and a variety of tissues within those strains (Cherbas and Cherbas, 1998). Traditionally, regulated gene expression in Drosophila cultured cell lines has been achieved by placing coding sequences of interest under direct control of a constitutive promoter, such as pAct5C from the actin5C gene (eg, Krasnow et al., 1989), or an inducible promoter, such as pMT from the metallothionein (mt) gene (Bunch et al., 1988), to create “direct-drive” systems. Because endogenous mt gene expression is the result of a systemic response to heavy metal toxicity in Drosophila (Maroni et al., 1987), inducible mt gene expression can be achieved by the introduction of heavy metals, normally copper ion (Cu) in the form of copper sulfate, into transiently or stably transfected cell lines (Bunch et al., 1988). The first approach to adapting the GAL4/UAS system for use in Drosophila cultured cell lines involved cotransfection of constructs that encoded specific proteins of interest under control of the GAL4-responsive UAS cassette with a constitutive GAL4 driver (eg, under control of the armadillo promoter, our unpublished results, or the act5C promoter, Johnson et al., 2000) to create a more flexible “indirect-drive” system. We have taken this system another step forward, introducing inducibility by placing GAL4 expression under control of the mt promoter in the widely used vector pRMHa-3 (Bunch et al., 1988;“pMT” hereafter). Through the use of this pMT: GAL4 construct and constructs that encode components of the Drosophila Notch (reviewed in Baron et al., 2002) and EGF receptor (dEGFR1, reviewed in Klambt, 2000) signal transduction pathways, we demonstrate that the GAL4/UAS system can be employed to craft inducible protein expression in Drosophila cell lines. This approach also benefits from the growing library of pUAST responder constructs available for the engineering of regulated gene expression in Drosophila. The use of an inducible GAL4/UAS system in cultured cells will provide control over the levels and timing of expression, allowing for lower expression of proteins that may cause toxicity when constitutively expressed. We used expression constructs encoding the wildtype Delta protein (DeltaWT; Kopczynski et al., 1988) and a constitutively active form of the Notch protein (NotchICD; Fortini et al., 1993) to compare the efficacy of the “indirect-drive” pMT: GAL4/pUAST system we have developed with that of the “direct-drive” pMT expression system (Bunch et al., 1988). The pMT: DeltaWT and pMT: NotchICD constructs have been used extensively in Drosophila cultured cells to study Delta–Notch interactions and downstream signaling events (Fehon et al., 1990; Klueg et al., 1998; Klueg and Muskavitch, 1999; Lieber et al., 2002). The pMT: DeltaWT or pMT: GAL4 and pUAST: DeltaWT constructs were transfected into Drosophila S3 cells, continuously induced, and levels of protein expression were assayed over time by Western blot analysis (Fig. 1). We found that when equimolar amounts of Delta-encoding constructs were used, expression levels driven by pMT: DeltaWT …