Name: functional cloning using a xenopus oocyte expression system
Uploaded: 2016-01-30T14:00:00
Description: Watch this Scientific Journal Video about Functional Cloning Using a Xenopus Oocyte Expression System at JoVE.com

This work was supported by a Professional Development Grant to C.Z.P. from the Shepherd University Foundation. The authors wish to thank Brett Zirkle and Malia Deshotel for helpful discussions on the protocols, and Dr. Carol Hurney for generous assistance.

Все экспериментальные процедуры были одобрены Университета Вирджинии Уходу за животными и использованию комитета. Примечание: на фиг.1 показан схематический обзор экспериментальных процедур. 1. Подготовка ооцитов <ol><li> Предварительн?...

<ol>
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Using Transposon-Mediated Insertional Mutagenesis.</a> Methods in Molecular Biology. 917, 111-127 (2012).</li><li>Grainger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+tropicalis.+as+a+Model+Organism+for+Genetics+and+Genomics:+Past,+Present+and+Future.">Xenopus tropicalis. as a Model Organism for Genetics and Genomics: Past, Present and Future.</a> Methods in Molecular Biology. 917, 3-15 (2012).</li><li>Nakayama, T., Fish, M., Fisher, M., Oomen-Hajagos, J., Thomsen, G., Grainger, R. <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+Efficient+CRISPR/Cas9-Mediated+Targeted+Mutagenesis+in+Xenopus+tropicalis.">Simple and Efficient CRISPR/Cas9-Mediated Targeted Mutagenesis in Xenopus tropicalis.</a> Genesis. 51, 835-843 (2013).</li><li>Ishibashi, S., Cliffe, R., Amaya, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+bi-allelic+mutation+rates+using+TALENs+in+Xenopus+tropicalis.">Highly efficient bi-allelic mutation rates using TALENs in Xenopus tropicalis.</a> Biology Open. 1 (12), 1273-1276 (2012).</li><li>Zhao, Y., Ishibashi, S., Amaya, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+Genetic+Studies+Using+Antisense+Morpholino+Oligonucleotides.">Reverse Genetic Studies Using Antisense Morpholino Oligonucleotides.</a> Methods in Molecular Biology. 917, 143-154 (2012).</li><li>Smith, W., Harland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injected+Xwnt-8.+RNA+acts+early+in+Xenopus.+embryos+to+promote+formation+of+a+vegetal+dorsalizing+center.">Injected Xwnt-8. 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Способ, описанный здесь, для функционального клонирования генов, способных индуцировать ответ в компетентный эктодермы может быть использован, чтобы идентифицировать широкий спектр продуктов генов. Этот метод расширяет прошлой работе путем объединения ткани вызывающие анализы мето...

Forward genetic approaches to identify genes of interest through their function or loss-of-function1,2 are an integral part of understanding complex patterning events in development. Coupled with powerful reverse genetic techniques available to an ever-widening array of systems and researchers3-5, it is now possible to identify genes with a key functional role in a pathway and then elucidate that function at the cellular level and in interaction with other gene products. One approach to functionally identifying genes of interest that has yielded many key findings in the past is expression cloning6,7.
Our recent aim8 was to identify early lens-inductive factors, since it has been demonstrated that initial steps in the vertebrate lens-inductive process occur as early as gastrula stages. To that end, we used the transiently lens-competent9 animal cap ectoderm (stage 11-11.510) of Xenopus embryos as responding tissue for induction, and the stage VI Xenopus oocyte as a source of production for the inducing factors.
The following protocol builds on the expression cloning and sib selection protocols of Smith and Harland6,7, also successfully used by others11-13. In our oocyte expression system (first utilized for production of inducing factors by Lustig and Kirschner14), pools of injected transcripts capable of directly or indirectly causing the oocytes to produce factors that elicit a lens-inductive response in animal cap ectoderm are selected for and identified. Since the system is useful for expressing secreted inducing molecules directly (oocyte-injected INHBB mRNA causes mesoderm induction in mesoderm-competent animal cap ectoderm8), we originally expected the screening procedure to be useful chiefly for identification of paracrine factors. However, since we identified a nuclear factor in our screen (ldb18), it is clear that the system can be used to identify a wide variety of molecules such as transcriptional or translational regulatory factors, miRNAs, cofactors, or juxtacrine factors.

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			<td height="42" style="height:42px;width:207px;">12/101 Antibody</td>
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			<td height="21" style="height:21px;width:207px;">Acetic anhydride</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">A6404</td>
			<td style="width:233px;">for ISH</td>
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			<td height="21" style="height:21px;width:207px;">Anti-Dig-AP</td>
			<td style="width:191px;">Roche</td>
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			<td height="21" style="height:21px;width:207px;">Aurum Plasmid Mini Kit</td>
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			<td height="21" style="height:21px;width:207px;">Blocking Reagent</td>
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			<td style="width:135px;">11096176001</td>
			<td style="width:233px;">for ISH</td>
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			<td height="21" style="height:21px;width:207px;">BM Purple</td>
			<td style="width:191px;">Roche</td>
			<td style="width:135px;">11442074001</td>
			<td style="width:233px;">for ISH</td>
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			<td height="21" style="height:21px;width:207px;">Boekel Hybridization Oven</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:135px;">13-245-121</td>
			<td style="width:233px;">for ISH</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Bouin&#39;s Solution</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">HT10132</td>
			<td style="width:233px;">for ISH</td>
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			<td height="21" style="height:21px;width:207px;">BSA</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">A9647</td>
			<td style="width:233px;">for OCM</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:207px;">CHAPS</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">C3023</td>
			<td style="width:233px;">for ISH</td>
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			<td height="21" style="height:21px;width:207px;">Collagenase A</td>
			<td style="width:191px;">Roche</td>
			<td style="width:135px;">10103578001</td>
			<td style="width:233px;">Defolliculation of oocytes</td>
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			<td height="21" style="height:21px;width:207px;">Cysteine</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">C121800</td>
			<td style="width:233px;">Dejelly embryos</td>
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			<td height="21" style="height:21px;width:207px;">DEPC-H2O</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:135px;">BP5611</td>
			<td style="width:233px;">for ISH</td>
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			<td height="21" style="height:21px;width:207px;">Dig-RNA Labeling Mix</td>
			<td style="width:191px;">Roche</td>
			<td style="width:135px;">11277073910</td>
			<td style="width:233px;">for ISH probes</td>
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			<td height="42" style="height:42px;width:207px;">Dumont #5 forceps</td>
			<td style="width:191px;">World Precision Instruments</td>
			<td style="width:135px;">500233</td>
			<td style="width:233px;">for Vitelline envelope removal</td>
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			<td height="21" style="height:21px;width:207px;">Ethyl 3-aminobenzoate</td>
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			<td style="width:233px;">for Injection media</td>
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			<td height="21" style="height:21px;width:207px;">Formamide</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">F9037</td>
			<td style="width:233px;">for ISH</td>
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			<td height="21" style="height:21px;width:207px;">Gentamicin sulfate</td>
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			<td style="width:233px;">for OCM</td>
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			<td height="42" style="height:42px;width:207px;">Glass capillaries</td>
			<td style="width:191px;">World Precision Instruments</td>
			<td style="width:135px;">4878</td>
			<td style="width:233px;">3.5&#34; long, I,D, 0.530mm</td>
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			<td height="21" style="height:21px;width:207px;">Glass sample vials</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:135px;">06-408B</td>
			<td style="width:233px;">for ISH</td>
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			<td height="42" style="height:42px;width:207px;">Hair loop</td>
			<td style="width:191px;"></td>
			<td style="width:135px;"></td>
			<td style="width:233px;">Hair affixed in pasteur pipette for tissue manipulation</td>
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			<td height="21" style="height:21px;width:207px;">Heparin sodium salt</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">H4784</td>
			<td style="width:233px;">for ISH</td>
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			<td height="42" style="height:42px;width:207px;">Injector Nanoliter 2010</td>
			<td style="width:191px;">World Precision Instruments</td>
			<td style="width:135px;">Nanoliter 2010</td>
			<td style="width:233px;">Microprocessor-controlled microinjector</td>
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			<td height="21" style="height:21px;width:207px;">Instant Ocean</td>
			<td style="width:191px;">Carolina</td>
			<td style="width:135px;">972433</td>
			<td style="width:233px;">Aquarium Salt for frog recovery</td>
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			<td height="42" style="height:42px;width:207px;">IRBG XGC Xenopus verified full-length cam cDNA</td>
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			<td style="width:135px;">989_IRBG</td>
			<td style="width:233px;">cDNA library&#160;</td>
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			<td height="42" style="height:42px;width:207px;">LB Agar plates with 100 &#181;g/mL Ampicillin</td>
			<td style="width:191px;">Teknova</td>
			<td style="width:135px;">L5004</td>
			<td style="width:233px;">150mm pre-poured LB-Amp plates for sib selection</td>
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			<td height="63" style="height:63px;width:207px;">LB Luria Broth</td>
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			<td style="width:233px;">LB for collecting colonies in sib selection from plates and dilution of cultures</td>
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			<td height="42" style="height:42px;width:207px;">Magnetic mRNA Isolation Kit</td>
			<td style="width:191px;">New England BioLabs</td>
			<td style="width:135px;">S1550S</td>
			<td style="width:233px;">for isolation of poly(A)-enriched RNA</td>
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			<td height="21" style="height:21px;width:207px;">Maleic Acid</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">M0375</td>
			<td style="width:233px;">for ISH</td>
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			<td height="42" style="height:42px;width:207px;">Manual Microfil Micromanipulator</td>
			<td style="width:191px;">World Precision Instruments</td>
			<td style="width:135px;">M3310R</td>
			<td style="width:233px;">Manual micromanipulator</td>
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			<td height="21" style="height:21px;width:207px;">Nutating Mixer</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:135px;">22-363-152</td>
			<td style="width:233px;">Rocker for ISH</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Permoplast</td>
			<td style="width:191px;">Nasco</td>
			<td style="width:135px;">SB33495M</td>
			<td style="width:233px;">Clay for injection and dissection dishes</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Phosphate Buffered Saline</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">P5368</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">PMSG</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">G4877</td>
			<td style="width:233px;">to stimulate oocyte development</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Polyvinylpyrrolidone</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">PVP40</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Programmable Puller</td>
			<td style="width:191px;">World Precision Instruments</td>
			<td style="width:135px;">PUL-1000</td>
			<td style="width:233px;">Micropipette needle puller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Proteinase K</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">P6556</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">pTnT Vector</td>
			<td style="width:191px;">Promega</td>
			<td style="width:135px;">L5610</td>
			<td style="width:233px;">cDNA library construction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Riboprobe Combination System</td>
			<td style="width:191px;">Promega</td>
			<td style="width:135px;">P1450</td>
			<td style="width:233px;">in vitro transcription</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Superscript Full Length cDNA Library Construction Kit</td>
			<td style="width:191px;">Life Technologies</td>
			<td style="width:135px;">18248013</td>
			<td style="width:233px;">kit for cDNA library construction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Sutures, 3-0 silk</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:135px;">19-037-516</td>
			<td style="width:233px;">Suture thread and needle for post-oocyte removal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Torula RNA</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">R3629</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Triethanolamine</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">T1502</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Tween 20</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">P9416</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Universal RiboClone cDNA Synthesis System</td>
			<td style="width:191px;">Promega</td>
			<td style="width:135px;">C4360</td>
			<td style="width:233px;">alternative kit for cDNA library construction</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:207px;">Xenopus Full ORF Entry Clones - ORFeome Collaboration</td>
			<td style="width:191px;">Source Bioscience</td>
			<td style="width:135px;">5055_XenORFeome</td>
			<td style="width:233px;">ORFeome Clones</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">XL2-Blue Ultracompetent Cells</td>
			<td style="width:191px;">Agilent Technologies</td>
			<td style="width:135px;">200150</td>
			<td style="width:233px;">cells for transformation of cDNA library</td>
		</tr>
	</tbody>
</table>

functional cloning using a xenopus oocyte expression system

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

В ответ на экспрессию мРНК введенного в ооцитах, отвечая животных колпачок ткань анализировали на экспрессию Otx2 от гибридизация (рисунок 2 и таблица 1); Otx2 выражается в предполагаемый линзы эктодермы (PLE) от закрытия нервной трубки через...

Watch this Scientific Journal Video about Functional Cloning Using a Xenopus Oocyte Expression System at JoVE.com

Functional Cloning Using a Xenopus Oocyte Expression System

Функциональная Клонирование Использование<em&gt; Xenopus</em&gt; Ооцитов выражение системы

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

Research

JoVE Journal

Developmental Biology

Functional Cloning Using a Xenopus Oocyte Expression System

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Понимание начале Органогенез, используя упрощенную<em&gt; На месте</em&gt; Гибридизация Протокола в<em&gt; Xenopus</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Живая съемка врожденной иммунной и предраковых межклеточного взаимодействия, используя / UAS Expression System Индуцибельная Gal4 на личиночной данио рерио кожи

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Альгинат инкапсуляции плюрипотентных стволовых клеток, используя коаксиальный Насадка

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

Использование конфокальной Анализ<em&gt; Xenopus Laevis</em&gt; По расследованию модуляторы Wnt и Shh морфогена градиентов

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

Отслеживание клеток в GFP-трансгенной данио рерио Использование системы Photoconvertible PSmOrange

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

Temporal Упорядочение динамических данных выражений из конкретных пространственных Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Функциональная Манипуляция материнских генных продуктов с помощью<em&gt; In Vitro</em&gt; Созревание ооцитов в данио рерио

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

Плода мышь сердечнососудистой визуализации с помощью системы высокой частоты ультразвука (30/45 МГц)

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Трехмерная система ассеаа ассеа ангиогенеза с использованием сокультурных сфероидов, сформированных эндотелиальными колоний формирования клеток и мезенхимальных стволовых клеток

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay

Определение программы трансляции материнской мРНК при созревании in vitro с помощью анализа одного репортера ооцитов

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that ...