Targeted and Conditional Gene Expression Workshop, 8th International Conference on Zebrafish Development and Genetics.
Targeted and Conditional Gene Expression Workshop, 8th International Conference on Zebrafish Development and Genetics.
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定向和条件基因表达研讨会，第八届斑马鱼发育和遗传学国际会议。
DOI：
10.1089/zeb.2008.0543
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发表时间：
2008
期刊：
影响因子：
2
通讯作者：
Hurlstone,AdamF
中科院分区：
文献类型：
--
作者：
Chen,JamesK;Hurlstone,AdamF
靶向和条件基因表达研讨会讨论了研究斑马鱼胚胎、幼虫和成虫基因功能的几种技术。该研讨会包括六次口头演讲，随后是对当前方法、其局限性和可能的解决方案的社区讨论。研究报告的重点是通过遗传和/或化学策略控制内源或外源基因活性的新方法。有条件调节内源基因功能的方法包括短发夹 RNA (shRNA) 表达、可逆转座子诱导的突变和笼状吗啉。控制外源基因活性的技术包括基于化学诱导孕酮受体的反式激活因子、Cre-LoxP 系统和雌激素受体融合蛋白。还讨论了在斑马鱼中使用 Gal4/UAS 系统的一般注意事项。 Adam Hurlstone 通过报告他的实验室对斑马鱼胚胎中 shRNA 介导的基因沉默的研究拉开了研讨会的序幕。与短干扰 RNA 相比，表达的 shRNA 似乎不会引起非特异性发育缺陷，Hurlstone 实验室报告称，他们通过向转基因胚胎注射 shRNA 表达构建体，成功阻断了转基因胚胎中增强型绿色荧光蛋白 (EGFP) 的表达。他们还使用 Fugutyrp1 启动子驱动的 shRNA 构建体瞬时抑制内源性酪氨酸酶表达和色素形成。目前正在努力培育能够以组织特异性和潜在可逆方式稳定表达 shRNA 的转基因斑马鱼。到目前为止，他们的初步研究表明shRNA表达水平必须达到一定的阈值才能实现有效的内源基因沉默，可能需要针对给定目标的扩增策略，例如Gal4/UAS系统或多个串联shRNA。Steve Ekker描述了一种用于可逆阻断斑马鱼内源基因功能的互补遗传方法。他的实验室创造了“基因断裂”转座子诱导的突变，其中侧翼为 LoxP 位点的剪接受体和供体位点通过转座子介导的整合插入到基因组 DNA 中。插入构建体还包含红色荧光蛋白 (RFP) 和 EGFP 编码序列，当在 RNA 成熟过程中使用异源剪接位点时，它们会生成包含 RFP 的截短基因产物和 EGFP 报告基因。使用这种方法，Ekker 实验室启动了对斑马鱼突变体的筛选，这些突变体表现出
Several technologies for studying gene function in zebrafish embryos, larvae, and adults were discussed at the Targeted and Conditional Gene Expression workshop. The workshop included six oral presentations followed by a community discussion of current approaches, their limitations, and possible solutions. The research talks focused on new methods for controlling the activity of endogenous or exogenous genes through genetic and/or chemical strategies. Approaches for conditionally regulating endogenous gene function included short-hairpin RNA (shRNA) expression, reversible transposon-induced mutations, and caged morpholinos. Technologies for controlling exogenous gene activity included a chemically inducible progesterone receptorbased transactivator, the Cre-LoxP system, and estrogen receptor fusion proteins. General considerations for using the Gal4/UAS system in zebrafish were also discussed. Adam Hurlstone kicked off the workshop by reporting his laboratory’s studies of shRNA-mediated gene silencing in zebrafish embryos. In contrast to short-interfering RNAs, expressed shRNAs do not appear to cause nonspecific developmental defects, and the Hurlstone laboratory reported that they have successfully blocked enhanced green fluorescent protein (EGFP) expression in transgenic embryos by injecting them with shRNA expression constructs. They have also transiently inhibited endogenous tyrosinase expression and pigment formation using an shRNA construct driven by the Fugu tyrp1 promoter. Efforts to generate transgenic zebrafish that stably express shRNAs in a tissue-specific and potentially reversible manner are underway. Thus far, their preliminary studies suggest that shRNA expression levels must reach a certain threshold to convey efficient endogenous gene silencing, perhaps necessitating amplification strategies such as the Gal4/UAS system or multiple, tandem shRNAs against a given target.Steve Ekker described a complementary genetic approach for reversibly blocking endogenous gene function in zebrafish. His laboratory has created ‘‘gene-break’’transposon-induced mutations, in which splice acceptor and donor sites flanked by LoxP sites are inserted into genomic DNA through transposon-mediated integration. The insertional constructs also contain red fluorescent protein (RFP) and EGFP coding sequences that generate an RFP-containing truncated gene product and EGFP reporter when the heterologous splice sites are used during RNA maturation. Using this approach, the Ekker laboratory has initiated a screen for zebrafish mutants that exhibit