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.

All experimental procedures were approved by the University of Virginia Institutional Animal Care and Use Committee.
Note: Figure 1 shows a schematic overview of the experimental procedures.
1. Preparation of Oocytes
<ol>
	<li>Pre-prime X. laevis females with 150 U of Pregnant Mare Serum Gonadotropin (PMSG) approximately one week in advance of oocyte isolation. Inject 1 ml 150 U/ml PMSG into dorsal lymph sac with 1 cc ste...

<ol>
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The method described here for the functional cloning of genes capable of inducing a response in competent ectoderm can be used to identify a wide range of gene products. This method expands upon past work by combining tissue-inducing assays with expression cloning techniques. We utilize the metabolic pathways of the Xenopus oocyte as a source of production of inducing factors, directly or indirectly, following RNA injection. This, in combination with the use of established methods for cloning a gene of interest<...

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;">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.

In response to expression of mRNA injected into oocytes, responding animal cap tissue was assayed for expression of otx2 by in situ hybridization (Figure 2 and Table 1); otx2 is expressed in the presumptive lens ectoderm (PLE) from neural tube closure through lens placode thickening19. However, since otx2 is also expressed in the anterior neural ectoderm as well as non-neural head ectoderm outside the PLE, it is associated wi...

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

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.

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 ...

The overall goal of this procedure is to demonstrate a Xenopus oocyte, an animal cap system, suitable for the identification of gene products capable of inducing a response in competent ectoderm. This method can help answer key questions in the field of developmental biology such as what genes are necessary to illicit an inductive response. The main advantage of this technique is its use of the Xenopus oocyte, as an expression system, to produce and identify inducers acting upon a target tissue, or even genes acting upstream of inducers.Visual demonstration of this method is extremely useful, as the dissection and recombination steps are highly stage dependent and require a refined technique. After collecting ovarian tissue from anesthetized female frogs, tear the tissue into small pieces, each containing approximately 10 to 20 oocytes. And transfer these fragments, first to fresh OR2-solution, and then to OR2-containing 2.0 milligrams per milliliter collagenase A.Continue by gently agitating the tissue-containing mixture on a shaker for one hour.Then transfer the fragments to fresh collagenase solution, and agitate for an additional hour. Expect to observe deflocculated oocytes at the end of this treatment. Proceed to wash the oocytes 10 times in complete OR2, and then twice in oocyte culture medium, abbreviated as OCM.Afterwards, place the oocytes in fresh OCM, and visually inspect them under a dissection microscope. Isolate those at stage six by discarding the smaller, immature oocytes. Next, place the oocytes in an agarose-coated Petri dish containing OCM, and maintain them at 18 to 20 degrees Celsius until injection.To prepare for microinjection, place glass capillary tubing into a needle puller, pull it, and then break the tips off the glass to generate needles approximately 20 micrometers in diameter. On a compound microscope, measure the needle tips with a calibrated ocular micrometer. Next, push clay into a uniform layer on the bottom of a 35 by 10 millimeter Petri dish.And then create parallel grooves in this layer, using a mall probe-like tool. Fill the clay-lined dish with approximately 2 milliliters of 3%Ficoll in 1x modified Barth's saline, or MBS. And then transfer the oocytes to it, using a wide-bore pipette.Position the oocytes in the groves so they will be held in place during the subsequent microinjection. Using a microinjector, fill a needle with approximately two microliters of a previously generated pool of mRNA and adjust the balance to produce slight positive pressure, which will prevent oocyte cytoplasm from being drawn into the needle during injection. Then, inject each oocyte with 20 nanoliters of RNA in the equatorial region.Afterwards, leave the oocytes in the Ficoll MBS for one hour, and then gently transfer them to 1x MBS. Proceed to incubate the oocytes for eight to 24 hours at 20 degrees Celsius. To begin the animal cap assay, gather 3/4x normal amphibian medium, abbreviated as NAM solution, fine forceps, a hairloop, previously injected oocytes, and xenopus embryos at stage 11 to 11.5.Then, break apart glass cover slips into fragments approximately one millimeters by two millimeters in size, and pass these pieces through a flame until their edges polish and drew. Next, press clay into a single layer in a dish, as previously described. And use a mall probe to create individual cup-shaped impressions in this coating.Proceed to fill the dish with approximately two milliliters of 3/4x NAM. And to it, transfer the injected oocytes, positioning several so that each is immobilized in a single indentation. To ready the embryos, transfer them in a dish containing 3/4x NAM solution.Select a subset of gastrula to be used as staging controls for the age of the ectoderm, and transfer them to another dish containing the same solution. And set this plate aside. For embryos that will be used in the animal cap assey, remove their vitelline membranes with two pairs of fine forceps.Afterwards, transfer the gastrula to the clay-lined dish with the oocytes. To begin, cut the animal caps from the embryos using two pairs of fine foreceps, being careful to avoid the equatorial tissue. To generate recombinants using the first method, position an animal caps so that the inner surface contacts the animal hemisphere of an oocyte, already positioned in an indentation.And repeat this process for several of the injected oocytes. To ensure that these recombinants are held together, place a curved glass coverslip fragment on top of each of them, and apply downward pressure until the animal is flattened and the coverslip contacts the clay. To generate recombinants using the second method, place animal caps and oocytes together in empty individual indentations in the clay.And ensure the animal caps inner surfaces are facing the oocyte. Then, secure the two tissues together using small extensions of clay. Once multiple recombinants have been generated using either method, culture then and the set-aside control embryos at 20 degrees celsius.When the control embryos reach the desired stage, remove the coverslips from the recombinants and isolate the animal cap ectoderm using forceps and a hair loop. Proceed to fix both ectodermal fragments and control embryos in MEMFA for one hour. And transfer them to ethanol and store this tissue at negative 20 degrees celsius.Here, sets of oocytes were injected with pools of mRNA corresponding to progressively smaller numbers of clones or colonies, and then, cultured with animal caps. Subsequently, the number of animal caps in each set expressing foxe3, a marker normally observed in presumptive lens ectoderm from early neural plate stages to vesicle formation, and use here, to evaluate lens inducing capability, was determined. This method identified in nuclear factor encoding gene, ldb1, which can produce a lens-inducing response in 50 out of 179, or 28%of animal caps assessed.Shown here, our in situ hybridization images, depicting foxe3 signal, indicated by arrow heads, in animal caps cultured with ldb1 injected oocytes from roughly stage 11 to stage 23. No foxe3 expression is observed in corresponding control animal caps placed on uninjected oocytes. When sections were generated from these animal caps, foxe3 expression was observed in both the inner and outer ectodermal layers.Expression in the inner layer is characteristic of a lens response. Or, as signaled in both layers, is indicative of other structures such as the olfactory placode. Following this procedure, methods like immunohistochemistry, in situ hybridization, or direct detection of a fluorescent reporter gene, can be performed in order to assess an inductive response in the animal cap ectoderm.

Research

JoVE Journal

Developmental Biology

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

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

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

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

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

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

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

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

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

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

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

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