Name: recombineering homologous recombination constructs in drosophila
Uploaded: 2013-07-13T12:00:00
Description: Watch this Scientific Journal Video about Recombineering Homologous Recombination Constructs in Drosophila at JoVE.com

我们想感谢雨果韶远试剂和布卢明顿库存中心。我们还要感谢公园Venken韶远，雨果和所有成员的Buszczak Hiesinger实验室的有益讨论。支持这项工作是由美国国家卫生研究院ACR（T32GM083831）的，公屋（RO1EY018884）的赠款和MB（RO1GM086647），授予德州癌症预防研究所以MB和公屋（RP100516），韦尔奇基金会（I-1657）公屋。 MB是在生物医学研究和公屋EE和格里尔加森•弗格森学者尤金·麦克德莫特学者在得克萨斯大学西南医学中心生物医学研究中。

1。选型的BAC和地区的目标<ol><li>要收购的BAC感兴趣的基因（GOI）（这里CG32095作为一个例子），搜索CG32095 <a href="http://www.flybase.org" target="_blank">www.flybase.org的</a> 。在部分股票和试剂，检查一节题为“基因组克隆”BAC的含CG32095。确保BAC包括至少10 KB的上游和下游5 kb的感兴趣的基因。或者，也可以被发现在P [ACMAN]载体7的克隆在<a href="http://www.pacmanfly.org/libraries.html" target=...

<ol>
	<li>Rong, Y. S., Golic, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+targeted+gene+knockout+in+Drosophila.">A targeted gene knockout in Drosophila.</a> Genetics. 157, 1307-1312 (2001).</li><li>Rong, Y. S., Golic, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+targeting+by+homologous+recombination+in+Drosophila.">Gene targeting by homologous recombination in Drosophila.</a> Science. 288, 2013-2018 (2000).</li><li>Gong, W. J., Golic, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ends-out,+or+replacement,+gene+targeting+in+Drosophila.">Ends-out, or replacement, gene targeting in Drosophila.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 2556-2561 (1073).</li><li>Warming, S., Costantino, N., Court, D. L., Jenkins, N. A., Copeland, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+highly+efficient+BAC+recombineering+using+galK+selection.">Simple and highly efficient BAC recombineering using galK selection.</a> Nucleic Acids Res. 33, e36 (2005).</li><li>Copeland, N. G., Jenkins, N. A., Court, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombineering:+a+powerful+new+tool+for+mouse+functional+genomics.">Recombineering: a powerful new tool for mouse functional genomics.</a> Nat. Rev. Genet. 2, 769-779 (2001).</li><li>Venken, K. J., He, Y., Hoskins, R. A., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P[acman]:+a+BAC+transgenic+platform+for+targeted+insertion+of+large+DNA+fragments+in+D.+melanogaster.">P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster.</a> Science. 314, 1747-1751 (2006).</li><li>Venken, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+P[acman]+BAC+libraries+for+transgenesis+studies+in+Drosophila+melanogaster.">Versatile P[acman] BAC libraries for transgenesis studies in Drosophila melanogaster.</a> Nat. Methods. 6, 431-434 (2009).</li><li>Groth, A. C., Fish, M., Nusse, R., Calos, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+transgenic+Drosophila+by+using+the+site-specific+integrase+from+phage+phiC31.">Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31.</a> Genetics. 166, 1775-1782 (2004).</li><li>Bischof, J., Maeda, R. K., Hediger, M., Karch, F., Basler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+transgenesis+system+for+Drosophila+using+germ-line-specific+phiC31+integrases.">An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 3312-3317 (2007).</li><li>Chan, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+discovery+of+Rab+GTPases+with+synaptic+functions+in+Drosophila.">Systematic discovery of Rab GTPases with synaptic functions in Drosophila.</a> Current Biology: CB. 21, 1704-1715 (2011).</li><li>Chan, C. C., Scoggin, S., Hiesinger, P. R., Buszczak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+recombineering+and+ends-out+homologous+recombination+to+systematically+characterize+Drosophila+gene+families:+Rab+GTPases+as+a+case+study.">Combining recombineering and ends-out homologous recombination to systematically characterize Drosophila gene families: Rab GTPases as a case study.</a> Commun. Integr. Biol. 5, 179-183 (2012).</li><li>Wu, N., Matand, K., Kebede, B., Acquaah, G., Williams, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+DNA+electrotransformation+efficiency+in+Escherichia+coli+DH10B+electrocompetent+cells.">Enhancing DNA electrotransformation efficiency in Escherichia coli DH10B electrocompetent cells.</a> Electronic Journal of Biotechnology. 13, (2010).</li><li>Wang, S., Zhao, Y., Leiby, M., Zhu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+positive/negative+selection+scheme+for+precise+BAC+recombineering.">A new positive/negative selection scheme for precise BAC recombineering.</a> Mol. Biotechnol. 42, 110-116 (2009).</li><li>Venken, K. J., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenesis+upgrades+for+Drosophila+melanogaster.">Transgenesis upgrades for Drosophila melanogaster.</a> Development. 134, 3571-3584 (2007).</li><li>Dietzl, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+transgenic+RNAi+library+for+conditional+gene+inactivation+in+Drosophila.">A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.</a> Nature. 448, 151-156 (2007).</li></ol>

在很大程度上是基于遗传操作的工具，可用于在生物医学研究中的遗传模式生物的力量。小模型C.线虫和果蝇的特别允许在多细胞发育或功能完整的途径和基因家族牵连的廉价和快速的分子遗传分析。近年来出现了显着进步，工具开发，操纵基因在果蝇 14,15。例如，重组工程，它被广泛应用于在小鼠遗传学操纵Bac的DNA构建体，最近为果蝇适应，通过使用的P [ACMAN]...

清洁分子操纵单个基因在他们的内源性基因位点提供了一个非常宝贵的工具来研究真核细胞生物学相关的问题无数。 果蝇反向遗传技术产生损失的功能基因已被证明是具有挑战性的，直到和他的同事介绍Golic 体内基因靶向使用果蝇 1-3同源重组。他们证明，采用线性的DNA片段从一个集成的转基因构造，可以针对特定的基因位点。这种线性的“捐助”的DNA 在体内产生通过FRT-介导的重组（消费作为环状分子从染色体DNA）大范围核酸酶I-SCEI线性。虽然这种方法已成功地用于生成各种定义的病变，该技术并没有轻松扩展平行的许多基因的操纵，因为每个INDIVI双淘汰赛结构需要不同的定制设计。例如，无缝地操纵使用经典的限制性内切酶/结扎克隆或PCR，以及大小的限制，传统的在体内转化载体的大片段的DNA（&gt; 5 kb的） 在体外的困难往往会干扰同源重组靶向快速创建向量。为了克服这些限制，我们结合重组工程/转基因P [ACMAN系统，它允许子克隆和高达100 kb的DNA的转基因的端部从基因打靶方法建立一个有效的和相对快速的平台，使有利于果蝇基因打靶。 重组介导的基因工程（重组工程）是一个强大的同源重组克隆技术4,5。对比常规的限制性内切酶/连接酶克隆，重组工程并不限于序列或SIZE的操纵DNA。重组工程采用了特殊的大肠杆菌coli菌株，港口重组机制提供了一个有缺陷的λ噬菌体4。最近这种技术已被采用，用于在果蝇 6,7。重组工程在果蝇依赖于修改有条件扩增细菌人工染色体（BAC）载体称为P [ACMAN] 6,7。这个载体进行两个的复制起源：oriV的化学诱导后，其产生的高拷贝数的净化需要大量的DNA测序和胚胎注入和ORIS基础条件下，维持低拷贝数。此外，P [ACMAN]载体配有细菌附着（attB位点）的网站。 attB位点作为基板fC31整合酶介导的转基因在果蝇基因组8,9成预定着陆点，允许注册成立的大型DNA片段。 我们已经产生一个P [ACMAN]载体（以下简称为的P [ACMAN]-KO 1.0），可以用作靶向载体的端部出同源重组10,11。要纳入两端的基因打靶技术的系统中，我们加入了两个FRT和两个I-SCEI网站。我们还包括网关盒，在这个修改后的载体，以简化程序纳入到的P [ACMAN] KO 1.0同源臂。这提供了一种快速和简单的方法到目标载体引入任意感兴趣的基因组区域。在这个协议中，我们将描述如何设计的靶向载体，使用P [ACMAN]-KO 1.0，如何调动此载体在体内的内源基因座定位。对于此协议的目的，我们将使用的RFP /简卡带更换感兴趣的基因，但这个协议，可以使用各种含有抗生素选择标记盒。我们已经设计并成功应用于一组磁带基因置换和标记10,11。

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>SW102 Recombination competent bacteria</td>
			<td>NCI-Frederick</td>
			<td>Recombination Bacteria (SW102, SW105 and SW106)</td>
			<td><a href="http://ncifrederick.cancer.gov/research/brb/logon.aspx" target="_blank">http://ncifrederick.cancer.gov/research/brb/logon.aspx</a></td>
		</tr>
		<tr>
			<td>TransforMax EPI300 electrocopmpetent E. coli</td>
			<td>Epicentre</td>
			<td>EC300110</td>
			<td>Includes CopyControl induction solution</td>
		</tr>
		<tr>
			<td>PfuUltra II Fusion HS DNA Polymerase</td>
			<td>Aligent Technology Inc.</td>
			<td>600670</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BamHI-HF</td>
			<td>New England Biolabs</td>
			<td>R3136S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Zymoclean Gel DNA Recovery Kit</td>
			<td>Zymo Research</td>
			<td>D4001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Use Gateway BP ClonaseII Enzyme kit</td>
			<td>Invitrogen</td>
			<td>11789-020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>P[acman]KO1.010</td>
			<td>Buszczak and Hiesinger Labs</td>
			<td>Upon request</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>pENTR RFP-Kan11</td>
			<td>Buszczak and Hiesinger Labs</td>
			<td>Upon request</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Flystocks</td>
			<td>Bloomington stock center</td>
			<td>Stock numbers: 25680, 25679</td>
			<td>y1 w*/Dp(2;Y)G, P{hs-hid}Y; P{70FLP}11 P{70I-SceI}2B snaSco/CyO, P{hs-hid}4 
			y1 w*/Dp(2;Y)G, P{hs-hid}Y; P{70FLP}23 P{70I-SceI}4A/TM3, P{hs-hid}14, Sb1</td>
		</tr>
		<tr>
			<td>Electroporation machine</td>
			<td>Biorad GenePulser Xcell with PC module</td>
			<td>165-2662</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cuvettes</td>
			<td>Fisher Brand</td>
			<td>#FB101</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

recombineering homologous recombination constructs in drosophila

快速，大规模的内源性基因操纵技术 ​​的持续发展，将扩大利用果蝇作为一种遗传模式生物为人类疾病相关的研究。近年来，人们像同源重组和重组工程技术进步。然而，产生明确的的无效突变或标记内源性蛋白仍然是一个重大的努力，大多数基因。这里，我们描述和展示的技术使用基于重组工程的克隆的方法生成，可用于目标和操作在体内的内源基因位点的载体，具体而言，我们已经建立了三种技术的组合：（1）血液中酒精浓度的转基因/重组工程，（ 2）两端的同源重组（3）网关技术，提供了一个可靠，高效和灵活的方法来处理内源性基因位点的。在这个协议中，我们提供了一步一步的详细信息：（1）设计individua升载体，（2）如何克隆大片段的基因组DNA同源重组到载体，使用间隙维修，及（3）如何更换或标记感兴趣的基因，在这些载体中，使用了第二轮的重组工程。最后，我们也将提供如何调动这些录音带在体内产生的淘汰赛，或通过一个标记基因敲的协议。这些方法可以很容易地采用平行的多个目标，并提供一种装置，用于及时和有效的方式操纵果蝇基因组。

LA及RA同源臂的扩增产生500 bp的产品和国有企业的PCR反应应该产生一个1.0 kb的产品（第2.1-2.4; 图2）。 2.5节进行BP反应通常是非常有效率和细菌转化产品产量平均5-100菌落。几乎所有的殖民地PCR测试与检查显示出预期的PCR产物。 在第一轮的重组工程（第3节）期望得到消化的P改造后的20-40殖民地ACMAN]-KO-1.0 SW102细胞含有LA及RA武器。这些克隆中的40％至60％，将携带所需...

Watch this Scientific Journal Video about Recombineering Homologous Recombination Constructs in Drosophila at JoVE.com

Recombineering Homologous Recombination Constructs in Drosophila

同源重组技术大大推进<em&gt;果蝇</em遗传学通过分子精确的突变使创建。最近通过的重组工程允许一个操作大块的DNA，并将其转化为<em&gt;果蝇</em<sup&gt; 6</sup&gt;。这里介绍的方法，这些技术结合起来，以迅速产生大量的同源重组载体。

重组工程同源重组构建<em&gt;果蝇</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

Research

JoVE Journal

Biology

Recombineering Homologous Recombination Constructs in Drosophila

Wolbachia Bacterial Infection in Drosophila

在果蝇的Wolbachia细菌感染

科研

生物学

Fabrication of Myogenic Engineered Tissue Constructs

制作生肌工程的组织结构

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological Recording in the Drosophila Embryo

在电生理记录果蝇胚胎

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

电子显微镜对胚胎的制备果蝇胚胎心管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

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

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

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 ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

使用内源性野生型BRCA1基因的细胞表达，确定同源重组的BRCA1基因突变的影响

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Imaging Cell Shape Change in Living Drosophila Embryos

Imaging Cell Shape Change in Living Drosophila Embryos

影像生活细胞的形状变化果蝇胚胎

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Genetic Modification and Recombination of Salivary Gland Organ Cultures

基因改造和重组唾液的腺体器官培养

Branching  morphogenesis occurs during the development of many organs, and the embryonic  mouse submandibular gland (SMG) is a classical model for the ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

MBP基于标记策略的Mgm101重组蛋白的制备

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the ...