Name: a reverse genetic approach to test functional redundancy during embryogenesis
Uploaded: 2010-08-11T17:00:00
Description: Watch this Scientific Journal Video about A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis at JoVE.com

在编制本介绍我们感谢他们的帮助埃文斯实验室成员。原来，这里使用的morpholinos博士奥黛丽Holtzinger表示验证。 TE是由来自美国国立卫生研究院（HL064282 HL056182）的赠款支持。按照研究所动物护理和使用委员会规定的准则和法规威尔康乃尔医学院，进行动物实验。

我们的目标是测试规范心肌祖细胞的两个转录因子的功能冗余。由两个gata5和gata6相关基因编码的因素和我们使用的是斑马鱼的模型，以了解他们的相关功能。我们的战略是阻止基因的功能，使用morpholinos 2 。我们将一个或成受精卵基因注入morpholinos，并与来自鸡蛋注射morpholinos同时的胚胎胚胎的表型。为了简化表型的评价，我们使用携带基因对心肌细胞的GFP从鱼的卵。 <p cl...

<ol>
	<li>Heicklen-Klein, A., McReynolds, L. J., Evans, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+zebrafish+model+to+study+GATA+transcription+factors.">Using the zebrafish model to study GATA transcription factors.</a> Semin Cell Dev Biol. 16, 95-106 (2005).</li><li>Bill, B. R., Petzold, A. M., Clark, K. J., Schimmenti, L. A., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+primer+for+morpholino+use+in+zebrafish.">A primer for morpholino use in zebrafish.</a> Zebrafish. 6, 69-77 (2009).</li><li>Holtzinger, A., Evans, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gata5+and+Gata6+are+functionally+redundant+in+zebrafish+for+specification+of+cardiomyocytes.">Gata5 and Gata6 are functionally redundant in zebrafish for specification of cardiomyocytes.</a> Dev Biol. 312, 613-622 (2007).</li><li>Peterkin, T., Gibson, A., Patient, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redundancy+and+evolution+of+GATA+factor+requirements+in+development+of+the+myocardium.">Redundancy and evolution of GATA factor requirements in development of the myocardium.</a> Dev Biol. 311, 623-635 (2007).</li><li>Holtzinger, A., Evans, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gata4+regulates+the+formation+of+multiple+organs.">Gata4 regulates the formation of multiple organs.</a> Development. 132, 4005-4014 (2005).</li><li>Sorrentino, R. P., Gajewski, K. M., Schulz, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GATA+factors+in+Drosophila+heart+and+blood+cell+development.">GATA factors in Drosophila heart and blood cell development.</a> Semin Cell Dev Biol. 16, 107-116 (2005).</li></ol>

在这里描述的实验，我们结合两个morpholinos，每个单独产生不同表型的范围，并发现一个完全不同的表型时，他们一起合作注入。当然，我们需要摆在首位，做实验的理由。我们怀疑这两个基因可能为早期功能补偿对方在这种情况下心脏规范，。这是因为基因高度相关（相同的DNA序列结合的能力）和重叠在胚胎发育 5的模式表示。此外，在果蝇遗传实验表明，GATA转录因子应为心脏规...

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<td>gata5 splice site morpholino</td>
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<td>Genetools, LLC</td>
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<td>Genetools, LLC</td>
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a reverse genetic approach to test functional redundancy during embryogenesis

通常由胚胎发育过程中基因功能丧失功能的实验，例如，通过有针对性的突变在小鼠（淘汰赛）。在斑马鱼模型，有效的反向遗传技术已经开发使用微量注射特定基因的反义morpholinos。 Morpholinos目标，通过特定的碱基配对和阻止基因的功能mRNA的瞬时抑制翻译或数天的胚胎发育过程中的拼接（击倒）。然而，在脊椎动物，如鼠标或斑马鱼，一些基因的功能可以被遮蔽的损失进行补偿的另一种基因的存在，由于这些方法。这是基因姐姐在同一发展组织共同表达的基因的家庭尤其如此。在斑马鱼，补偿功能，可以在一个相对高通量的方式进行测试，共注射morpholinos目标击倒的这两个基因同时。同样，使用morpholinos，任何两个基因之间的遗传相互作用，可以证明这两个基因敲除在亚阈值水平。例如，morpholinos可滴定等，无论是个人击倒生成一个表型。下，如果这些条件中，双方morpholinos的合作注入导致的表型，是一种遗传性的互动。在这里，我们演示了如何显示在两个相关的转录因子GATA转录因子方面的功能冗余。转录因子GATA因素是必不可少的心脏祖细胞的规范，但是这仅仅是损失Gata5和Gata6透露。我们如何进行显微注射​​实验，验证morpholinos，评估补偿cardiogenesis表型。

Watch this Scientific Journal Video about A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis at JoVE.com

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

基因的功能，可以掩盖损失功能的实验中，如果有另一种基因的赔偿。斑马鱼模型提供了一个相对高通量的手段来揭示生活胚胎这样的功能冗余。

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Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

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