Name: mosaic zebrafish transgenesis for evaluating enhancer sequences
Uploaded: 2010-07-16T17:00:00
Description: Watch this Scientific Journal Video about Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences at JoVE.com

<p class="jove_content">我们感谢女士出色的鱼牧保罗伊，史蒂夫Ekker pDB600质粒礼物。这些研究是由来自NHGRI批到SF。 </p>

<p class="jove_title"> 1。选型和测试的保守序列的克隆</p><ol><li>首先必须找出潜在的调控序列进行功能测试。我们依赖于进化的序列保守性作为标准来选择序列，最经常使用的算法PhastCons，这是作为跟踪UCSC基因组浏览器（http://genome.ucsc.edu/cgi-bin/hgGateway）访问。然而，还有许多其他可用来评估跨物种的序列保守的算法。 </li><li>一旦我们选择了我们的序列，设计引物扩增基因组DNA的每个序列。应选择引物扩增保守的区域?...

<ol>
	<li>Fisher, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+biological+relevance+of+putative+enhancers+using+Tol2+transposon-mediated+transgenesis+in+zebrafish.">Evaluating the biological relevance of putative enhancers using Tol2 transposon-mediated transgenesis in zebrafish.</a> <em style='font-style: italic'>Nat Protoc</em>. <b>1</b> (3), 1297-12 (2006).</li><li>Balciunas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+a+high+cargo-capacity+transposon+for+genetic+applications+in+vertebrates.">Harnessing a high cargo-capacity transposon for genetic applications in vertebrates.</a> <em style='font-style: italic'>PLoS Genet</em>. <b>2</b> (11), e169-169 (2006).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>The Zebrafish Book</em>. , (1995).</li></ol>

<p class="jove_content">我们已经证明，在我们的实验室用于潜在的增强剂使用斑马鱼转基因的高效分析方法。大多数情况下，我们使用这种方法，寻找与人类基因相关的组织特异性的增强剂。尽管缺乏明显的序列同源性之间的人类和鱼类非编码序列，我们发现，这种方法通常是在成功地确定我们感兴趣的基因的增强剂。成功的关键因素，正在编制的质粒DNA和RNA的转座护理，注射和检查每个构造足够数量的胚胎。转基因?...

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<td>Takara LA Taq</td>
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<td>Takara Bio Inc</td>
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<td>Invitrogen</td>
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<td>Library efficiency competent cells DH5&alpha;</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12535-019</td>
<td>&nbsp;</td>
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<td>Library efficiency competent cells DB3.1</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
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<td>mMESSAGE mMACHINE Kit</td>
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<td>QIAquick PCR Purification Kit </td>
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<td>Qiagen</td>
<td>28104</td>
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<td>Flaming/Brown Micropipette Puller</td>
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<td>Sutter Instrument</td>
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<td>Pneumatic PicoPump</td>
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<td>World Precision Instruments</td>
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mosaic zebrafish transgenesis for evaluating enhancer sequences

<p class="jove_content">人类基因组序列的完成，以及其他许多物种，突出了把特定功能的非编码序列的挑战。由非编码的基因组的一部分进行的一个突出功能是调节基因的转录;但是，有没有有效的方法来大致预测的主要DNA序列的顺式调控元件。我们已经开发出一种高效的协议功能评估潜在的顺式调控，通过斑马鱼转基因的元素。我们的方法提供了发育的重要基因的细胞培养为基础的技术，显着的优势，因为它提供信息的空间和时间的基因调控。相反，它是更快和更昂贵的比类似的实验，在转基因小鼠，我们经常将其应用到人类基因组序列。在这里，我们证明我们的方法来测试序列克隆序列的养护和我们的协议的基础上，显微注射到斑马鱼的胚胎中选择元素。</p>

Watch this Scientific Journal Video about Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences at JoVE.com

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

<p class="jove_content">我们证明我们的方法来寻找潜在发育调控基因的增强子元件和评估它们的功能，通过镶嵌的斑马鱼转基因。</p>

马赛克斑马鱼转基因增强子序列，以评估

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non ...

Research

JoVE Journal

Biology

Zebrafish Brain Ventricle Injection

斑马鱼脑室注射液

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

科研

生物学

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode <em>Pristionchus pacificus</em>

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

通过显微注射介导的RNAi Necromenic线虫基因敲除和转基因<em> Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

<em>In vivo</em> Electroporation of Morpholinos into the Adult Zebrafish Retina

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

<em>在体内</em>电穿孔Morpholinos到成年斑马鱼视网膜

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

<em>In vivo</em> Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

再生的成年斑马鱼尾鳍<em>在</em>吗啉代入<em>体内</em>电穿孔

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

在成年斑马鱼的生化分析采血车

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

在斑马鱼发育的细胞追踪使用Photoconvertible蛋白

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

激光伤害的斑马鱼胚胎骨骼肌

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult <em>Drosophila</em> Photoreceptor Cells

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

番茄/ GFP-FLP / FRT方法马赛克成人的实时成像<em&gt;果蝇</em&gt;感光细胞

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

生精细胞类型使用STA把速度沉降分离

Mammalian spermatogenesis is a complex  differentiation process that occurs in several stages in the seminiferous  tubules of the testes. Currently, there ...

Automated Analysis of Dynamic Ca<sup>2+</sup> Signals in Image Sequences

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

动态钙的自动分析<sup&gt; 2 +</sup&gt;在图像序列信号

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...