Pogostick: A New Versatile piggyBac Vector for Inducible Gene Over-Expression and Down-Regulation in Emerging Model Systems
Pogostick: A New Versatile piggyBac Vector for Inducible Gene Over-Expression and Down-Regulation in Emerging Model Systems
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DOI：
10.1371/journal.pone.0018659
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发表时间：
2011-04-14
期刊：
影响因子：
3.7
通讯作者：
Monteiro, Antonia
中科院分区：
文献类型：
--
作者：
Chen, Bin;Hrycaj, Steven;Monteiro, Antonia
背景：非传统模型系统需要新的工具，使它们能够进入功能遗传学领域。这些工具应该能够通过敲除内源基因以及转基因的过度表达和异位表达来探索基因功能。方法：我们构建了一种名为Pogostick的新载体，它可以用于在生物中过度表达或下调基因，这些基因适合于通过iggyBac转座元件进行生殖系转化。Pogotick可以在www.addgene.org上找到，这是一个非营利性的质粒库。该载体目前使用来自果蝇的热休克启动子Hsp70来驱动转基因表达，因此将立即适用于能够正确解释该启动子序列的生物。我们详细介绍了如何将候选基因克隆到这个载体中，并通过靶向编码荧光蛋白DsRed的基因在果蝇中测试其功能。通过将单个DsRed拷贝克隆到载体中，并产生转基因株系，我们发现热休克后DsRed的mRNA和蛋白水平都有所提高。当反向克隆DsRed的第二个拷贝到侧翼位置，并将表达DsRed的果蝇结构性地转化到眼睛中时，我们发现热休克后内源mRNA和蛋白质水平下降。然后，我们在一个新兴的模型系统--Anyclus anynana中测试了包含Ubx基因完整cDNA的过表达载体。我们建立了一个转基因株系，并显示在热休克后Ubx mRNA的表达水平显著上升。最后，我们展示了如何获得Pogostick插入位点附近的基因组序列，以及如何估计转化个体基因组中的转基因拷贝数。意义：这种新的载体将允许新兴的模型系统以较少的障碍进入功能遗传学领域。
Background: Non-traditional model systems need new tools that will enable them to enter the field of functional genetics. These tools should enable the exploration of gene function, via knock-downs of endogenous genes, as well as over-expression and ectopic expression of transgenes.Methodology: We constructed a new vector called Pogostick that can be used to over-express or down-regulate genes in organisms amenable to germ line transformation by the piggyBac transposable element. Pogostick can be found at www.addgene.org, a non-profit plasmid repository. The vector currently uses the heat-shock promoter Hsp70 from Drosophila to drive transgene expression and, as such, will have immediate applicability to organisms that can correctly interpret this promotor sequence. We detail how to clone candidate genes into this vector and test its functionality in Drosophila by targeting a gene coding for the fluorescent protein DsRed. By cloning a single DsRed copy into the vector, and generating transgenic lines, we show that DsRed mRNA and protein levels are elevated following heat-shock. When cloning a second copy of DsRed in reverse orientation into a flanking site, and transforming flies constitutively expressing DsRed in the eyes, we show that endogenous mRNA and protein levels drop following heat-shock. We then test the over-expression vector, containing the complete cDNA of Ultrabithorax (Ubx) gene, in an emerging model system, Bicyclus anynana. We produce a transgenic line and show that levels of Ubx mRNA expression rise significantly following a heat-shock. Finally, we show how to obtain genomic sequence adjacent to the Pogostick insertion site and to estimate transgene copy number in genomes of transformed individuals.Significance: This new vector will allow emerging model systems to enter the field of functional genetics with few hurdles.