生产 C。线虫</em通过重组工程与转基因 galK选择标记
Summary
能力生产的转基因<em>线虫</em>使用fosmids进行基因组DNA作为保留所有原生的调控元件，是特别有吸引力。描述的是一个简单和强大的过程，通过生产转基因的重组工程<em> galK</em>选择标记。
Abstract
转基因动物的建立是广泛使用在 C 线虫的研究，包括研究有关规例及利益或代串联亲和纯化（TAP）的标签的特定基因的版本，以方便其净化的基因表达模式的使用GFP融合蛋白。典型的转基因是通过配售上游启动子的GFP报告基因或cDNA利益的产生，这往往产生一个代表性的表达模式。然而，基因调控的关键要素，如控制在3'非编码区或替代促销员元素，，可以错过这种方法。进一步只有一个单一的拼接变异体，通常可以通过，这意味着研究。相比之下，使用fosmid DNA克隆的蠕虫病毒基因组进行DNA的可能包括大多数，如果不是在基因调控在体内 ，它允许更大的能力，捕捉到真正的表达模式和时序所涉及的所有元素。为了方便使用fosmid DNA的转基因代，我们描述了一个E 。 大肠杆菌基于重组工程的程序，绿色荧光蛋白，TAP标签，或其他感兴趣的序列插入到任何基因的位置。该过程使用都在重组工程的正面和负面的选择步骤，高效率地获得所需的修改结果 galK基因作为选择标记。此外，包含galK基因两侧常用的绿色荧光蛋白的同源性武器和TAP融合基因的质粒可减少50％的成本寡核苷酸时产生的GFP或TAP融合蛋白。这些质粒使用R6K复制起点，这就排除了需要进行广泛的PCR产物纯化。最后，我们还展示了技术集成允许fosmid直接注射或轰击成蠕虫产生转基因动物的fosmid骨干UNC – 119标记。这个视频演示，通过使用这种方法重组工程生成转基因有关手续。
Protocol
概述许多转基因在转基因C.代线虫包括启动子序列，也许是基因的cDNA克隆到一个由博士安迪消防1实验室生成的向量。虽然这些转基因往往与生产GFP报告基因的cDNA表达所需的模式方面的成功，这些转基因缺乏备用的推动者，增强子元件，和3'非翻译区（UTR）元素发挥重要的作用，在控制在体内的基因表达。例如，无论是DAF – 12和FAH – 1…
Discussion
转基因从fosmids一代提供保留所有本机的启动子，剪接变异体，和3'非编码区调控元件的利益。这可能会导致一个转基因这是原生的表达模式，或建设功能的基因，当其他方法失败5反射的建设。由此产生的转基因可以进行各种包括GFP或自来水标记的抗原表位的标签。
转基因的建设涉及到三个步骤都进行SW106细菌染色 15 。首先，galK基因融入在fosmid所需?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者想感谢纳什与发展中国家的技术帮助林赛。这项工作是由美国国立卫生研究院授予AG028977 ALF的，从美国匹兹堡大学的一个OAIC匹兹堡大学（AG024827），种子基金的试点项目授予。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| FosmidMAX kit | Epicentre | FMAX046 | ||
| GoTaq | Promega | M7122 | ||
| MOPS Media | Teknova | M2120 | ||
| 0.132 M Potassium phosphate solution | Teknova | M2102 | ||
| D-galactose | Sigma | G0750 | ||
| 2-deoxygalactose | Sigma | D4407 | ||
| Biotin | Sigma | B4639 | ||
| Leucine | Sigma | L8000 | ||
| NH4Cl | Sigma | A9434 | ||
| Phusion DNA polymerase | NEB | F-530S | ||
| MacConkey agar base | Becton Dickinson | 281810 | ||
| Arabinose | Sigma | A3131 | ||
| Chloramphenicol | Sigma | C1919 | ||
| Sodium phosphate dibasic | Sigma | S5136 | ||
| Potassium phosphate monobasic | Sigma | P5655 | ||
| Sodium chloride | Sigma | S5886 | ||
| Glycerol | Sigma | G2025 | ||
| Bacto Agar | Becton Dickinson | 214010 |
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Tags
C. ElegansTransgenesRecombineeringGalK Selectable MarkerGFP Fusion ProteinsGene RegulationExpression PatternTandem Affinity Purification (TAP)PromoterReporter GeneCDNA3′ Untranslated RegionAlternative PromotersWorm Genomic DNAFosmid DNA ClonesE. Coli Based Recombineering ProcedureGFP InsertionTAP-tag InsertionPositive SelectionNegative Selection
Cite This Article
Zhang, Y., Kashyap, L., Ferguson, A. A., Fisher, A. L. The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker. J. Vis. Exp. (47), e2331, doi:10.3791/2331 (2011).