Transgenic tools for members of the genus Drosophila with sequenced genomes
Transgenic tools for members of the genus Drosophila with sequenced genomes
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DOI：
10.4161/fly.4.4.13304
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发表时间：
2010-10-01
期刊：
影响因子：
1.2
通讯作者：
Kaufman, Thomas
中科院分区：
文献类型：
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作者：
Holtzman, Stacy;Miller, David;Kaufman, Thomas
12种果蝇的基因组测序为这些物种之间的比较分子分析创造了很多机会。为了帮助这一努力，我们已经基于piggyBac转座子和3x P3-EGFP和-ECFP转基因标记制作了几种转化载体，这些转化载体应该有助于在这些物种中进行诱变和建立GAL4/UAS系统。我们已经测试了mini-white作为插入诱变标记的能力，并观察到在假毛囊D.和雄性D.的一个亚系中，睾丸鞘有mini-white衍生的色素沉着。我们已经将piggyBac转座酶的一个来源整合到9个果蝇物种中，并证明了这些转座酶系在体内动员标记插入物的功能。此外，我们还测试了一种黑腹d.a melanogaster纳米增强元件在黑腹d.a melanogaster、拟象d.a simulans、勃起d.a yakuba、伪眼d.a roobscura和雄性d.a virilis中驱动GAL4表达的能力。通过测量UAS-EGFPtub在所有6个物种中的反应来确定no - gal4转基因的有效性。我们的研究结果表明，在其他物种的卵巢、睾丸和胚胎中，在不同程度上，没有gal4驱动表达，就像在黑腹龙中看到的那样。然而，表达水平是可变的，这表明在某些情况下可能需要使用物种特异性启动子。总之，我们希望在这项工作的基础上，为插入诱变和基于GAL4/UAS系统的多物种果蝇实验提供一套指导方针和基本工具。
The sequencing of the genomes of 12 Drosophila species has created an opportunity for much in the way of comparative molecular analyses amongst these species. To aid that endeavor, we have made several transformation vectors based on the piggyBac transposon with 3x P3-EGFP and -ECFP transgenic markers that should be useful for mutagenesis and establishing the GAL4/UAS system in these species. We have tested the ability of mini-white to be used as a marker for insertional mutagenesis, and have observed mini-white-derived pigmentation of the testes sheath in a subset of lines from D. pseudoobscura and D. virilis. We have incorporated a source of piggyBac transposase into nine Drosophila species, and have demonstrated the functionality of these transposase lines for mobilization of marked inserts in vivo. Additionally, we tested the ability of a D. melanogaster nanos enhancer element to drive expression of GAL4 in D. melanogaster, D. simulans, D. erecta, D. yakuba, D. pseudoobscura and D. virilis. The efficacy of the nos-Gal4 transgene was determined by measuring the response of UAS-EGFPtub in all six species. Our results show that D. melanogaster nos-Gal4 drives expression in other species, to varying degrees, in similar spatiotemporal domains in the ovaries, testes and embryos as seen in D. melanogaster. However, expression levels are variable, demonstrating the possible need to use species-specific promoters in some cases. In summary, we hope to provide a set of guidelines and basic tools, based upon this work, for both insertional mutagenesis and GAL4/UAS system-based experiments in multiple species of Drosophila.