Name: the production of c. elegans transgenes via recombineering with the galk selectable marker
Uploaded: 2011-01-11T23:58:00
Description: Watch this Scientific Journal Video about The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker at JoVE.com

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。

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&#39;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...

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W., Model, P., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homologous+recombination+based+modification+in+Escherichia+coli+and+germline+transmission+in+transgenic+mice+of+a+bacterial+artificial+chromosome.">Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome.</a> Nat Biotechnol. 15, 859-865 (1997).</li><li>Court, D. L., Sawitzke, J. A., Thomason, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+engineering+using+homologous+recombination.">Genetic engineering using homologous recombination.</a> Annu.Rev.Genet. 36, 361-388 (2002).</li><li>Westenberg, M., Bamps, S., Soedling, H., Hope, I. A., Dolphin, C. 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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&#39;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?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>FosmidMAX kit</td>
<td>&nbsp;</td>
<td>Epicentre</td>
<td>FMAX046</td>
<td>&nbsp;</td>
<tr>
<td>GoTaq</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>M7122</td>
<td>&nbsp;</td>
<tr>
<td>MOPS Media</td>
<td>&nbsp;</td>
<td>Teknova</td>
<td>M2120</td>
<td>&nbsp;</td>
<tr>
<td>0.132 M Potassium phosphate solution</td>
<td>&nbsp;</td>
<td>Teknova</td>
<td>M2102</td>
<td>&nbsp;</td>
<tr>
<td>D-galactose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G0750</td>
<td>&nbsp;</td>
<tr>
<td>2-deoxygalactose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D4407</td>
<td>&nbsp;</td>
<tr>
<td>Biotin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B4639</td>
<td>&nbsp;</td>
<tr>
<td>Leucine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L8000</td>
<td>&nbsp;</td>
<tr>
<td>NH4Cl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A9434</td>
<td>&nbsp;</td>
<tr>
<td>Phusion DNA polymerase</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>F-530S</td>
<td>&nbsp;</td>
<tr>
<td>MacConkey agar base</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>281810</td>
<td>&nbsp;</td>
<tr>
<td>Arabinose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A3131</td>
<td>&nbsp;</td>
<tr>
<td>Chloramphenicol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C1919</td>
<td>&nbsp;</td>
<tr>
<td>Sodium phosphate dibasic</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S5136</td>
<td>&nbsp;</td>
<tr>
<td>Potassium phosphate monobasic</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5655</td>
<td>&nbsp;</td>
<tr>
<td>Sodium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S5886</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G2025</td>
<td>&nbsp;</td>
<tr>
<td>Bacto Agar</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>214010</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

the production of c. elegans transgenes via recombineering with the galk selectable marker

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&#39;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。

Watch this Scientific Journal Video about The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker at JoVE.com

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

能力生产的转基因<em&gt;线虫</em&gt;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&gt; galK</em&gt;选择标记。

生产 C。线虫</em通过重组工程与转基因 galK选择标记

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

Research

JoVE Journal

Biology

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

Generation of Stable Transgenic C. elegans Using Microinjection

产生稳定的转基因C.使用微量注射线虫

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

科研

生物学

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

一个线虫多巴胺神经元退化含量的应用验证潜在的帕金森氏症基因

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

Dissecting and Recording from The C. Elegans Neuromuscular Junction

线虫神经肌肉交界处的解剖和录制

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

A Protocol for the Production of KLRG1 Tetramer

KLRG1四聚体的生产协议

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. ...

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

测量线虫寿命在96孔板

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

高通量筛选和生物传感与荧光 C。线虫菌株

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

创建定义的气体环境研究缺氧对<em&gt; C。线虫</em

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

使用屏幕在车身尺寸规例中的线虫基因介导的RNA干扰取食策略<em&gt; C.线虫</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

C. elegans Chemotaxis Assay

C. elegans Chemotaxis Assay

线虫趋含量的

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that ...

生产 C。线虫 galK选择标记