A toolkit and robust pipeline for the generation of fosmid-based reporter genes in C. elegans.
A toolkit and robust pipeline for the generation of fosmid-based reporter genes in C. elegans.
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DOI：
10.1371/journal.pone.0004625
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发表时间：
2009
期刊：
影响因子：
3.7
通讯作者：
Hobert O
中科院分区：
文献类型：
--
作者：
Tursun B;Cochella L;Carrera I;Hobert O
将荧光蛋白工程化到包含在细菌人工染色体（BACs）或福斯质粒载体中的大型基因组克隆中，是一种在转基因动物中可视化和研究时空基因表达模式的工具。由于这些报告基因涵盖了较大的基因组区域，它们很可能捕获所有的顺式调控信息，因此有望重现内源基因表达的各个方面。通过同源重组（“重组工程”）在基因组克隆内所含的靶基因位点插入标签是生成这些报告基因的最直接方法。在这篇方法学论文中，我们描述了一个用于福斯质粒重组工程的简单且稳健的流程，我们将其应用于在线虫秀丽隐杆线虫中生成报告基因构建体，其基因组几乎完全被一个可用的福斯质粒文库所覆盖。我们已经构建了一个工具包，它能够以几乎无缝的方式在福斯质粒克隆内的特定靶点插入荧光蛋白（绿色荧光蛋白、黄色荧光蛋白、青色荧光蛋白、金星蛋白、樱桃红蛋白）和亲和标签。我们的新流程不像之前描述的用于生成秀丽隐杆线虫表达研究报告基因融合体的重组工程策略那么复杂，而且在我们的操作中更稳健。此外，我们的工具包提供了一种新型的重组盒，它在感兴趣的基因和荧光蛋白之间插入一个SL2剪接的顺反子间区域，从而创建一个由所研究基因的所有5′和3′顺式作用调控元件控制的报告基因，而两者之间没有直接的翻译融合。通过这种配置，可以很容易地检测到分泌型、亚细胞区室化或短寿命基因产物的表达起始和组织特异性。我们还描述了福斯质粒重组工程的其他应用。这里描述的重组工程流程的简单性、快速性和稳健性应该会促使这种策略在秀丽隐杆线虫表达研究中得到常规使用。
Engineering fluorescent proteins into large genomic clones, contained within BACs or fosmid vectors, is a tool to visualize and study spatiotemporal gene expression patterns in transgenic animals. Because these reporters cover large genomic regions, they most likely capture all cis-regulatory information and can therefore be expected to recapitulate all aspects of endogenous gene expression. Inserting tags at the target gene locus contained within genomic clones by homologous recombination (“recombineering”) represents the most straightforward method to generate these reporters. In this methodology paper, we describe a simple and robust pipeline for recombineering of fosmids, which we apply to generate reporter constructs in the nematode C. elegans, whose genome is almost entirely covered in an available fosmid library. We have generated a toolkit that allows for insertion of fluorescent proteins (GFP, YFP, CFP, VENUS, mCherry) and affinity tags at specific target sites within fosmid clones in a virtually seamless manner. Our new pipeline is less complex and, in our hands, works more robustly than previously described recombineering strategies to generate reporter fusions for C. elegans expression studies. Furthermore, our toolkit provides a novel recombineering cassette which inserts a SL2-spliced intercistronic region between the gene of interest and the fluorescent protein, thus creating a reporter controlled by all 5′ and 3′ cis-acting regulatory elements of the examined gene without the direct translational fusion between the two. With this configuration, the onset of expression and tissue specificity of secreted, sub-cellular compartmentalized or short-lived gene products can be easily detected. We describe other applications of fosmid recombineering as well. The simplicity, speed and robustness of the recombineering pipeline described here should prompt the routine use of this strategy for expression studies in C. elegans.
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