Name: a cell-free assay using xenopus laevis embryo extracts to study mechanisms of nuclear size regulation
Uploaded: 2016-08-08T14:00:00
Description: Watch this Scientific Journal Video about A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation at JoVE.com

Members of the Levy and Gatlin labs as well as colleagues in the Department of Molecular Biology offered helpful advice and discussions. Rebecca Heald provided support in the early stages of developing this protocol. This work was supported by the NIH/NIGMS (R15GM106318) and the American Cancer Society (RSG-15-035-01-DDC).

All Xenopus procedures and studies were conducted in compliance with the NRC Guide for the Care and Use of Laboratory Animals 8th edition. Protocols were approved by the University of Wyoming Institutional Animal Care and Use Committee (Assurance # A-3216-01).
1. Preparation of X. laevis Egg Extract (adapted from27,28)
<ol>
	<li>Prime female&#160;X. laevis&#160;frogs a minimum of three days and a maximum of two weeks before egg collection with a single 100 IU injection of preg...

<ol>
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Here is presented a novel method to study mechanisms of nuclear size regulation during X. laevis development. Developmental progression is associated with dramatic changes in cell physiology, metabolism, division rates, and migration, as well as alterations in the sizes of cells and intracellular structures. These varied processes are complex and essential, so it is difficult to study just one of these aspects of development in an in vivo setting. The X. laevis embryo extract and nuclear shrink...

The sizes of cellular organelles typically scale with the size of the cell, and this has been perhaps best documented for the scaling of nuclear size with cell size1-10. This is particularly true during embryogenesis and cell differentiation, when dramatic reductions in both cell and nuclear size are often observed11,12. Furthermore, altered nuclear size is a key parameter in cancer diagnosis and prognosis13-17. Mechanisms that contribute to nuclear size regulation are largely unknown, in part due to the complexity and essential nature of nuclear structure and function. The method described here was developed as an in vitro assay for nuclear size scaling that is amenable to biochemical manipulation and elucidation of mechanisms of nuclear size regulation.
Xenopus laevis egg extract is a well-established system to recapitulate and study complex cellular processes in an in vitro context. These extracts have revealed new fundamental information about several cellular processes including the assembly and function of the mitotic spindle, endoplasmic reticulum, and nucleus18-22. One key advantage to the extract system is that X. laevis egg extracts represent a nearly undiluted cytoplasm whose composition can be easily altered, for instance through addition of recombinant proteins or immunodepletion. Furthermore, one is able to manipulate essential processes by employing treatments that might otherwise be lethal in an in vivo context. Modifications of the egg extract procedure allow for isolation of extracts from X. laevis embryos rather than eggs, and these embryo extracts are equally amenable to biochemical manipulation23. During X. laevis development, the single-cell fertilized embryo (~1 mm diameter) undergoes a series of twelve rapid cell divisions (stages 1 - 8) to generate several thousand 50 &#181;m diameter and smaller cells, reaching a developmental stage termed the midblastula transition (MBT) or stage 824-26. The MBT is characterized by the onset of zygotic transcription, cell migration, asynchronous cell divisions, acquisition of gap phases, and establishment of nuclear steady-state sizes rather than continual nuclear expansion as in the pre-MBT embryo. From stage 4 to gastrulation (stages 10.5 - 12), the volume of individual nuclei decreases by more than 10-fold11.
Here, the goal is to identify mechanisms responsible for these reductions in nuclear size during developmental progression. The approach is to first assemble nuclei in X. laevis egg extract and to isolate those nuclei from the egg cytoplasm/extract. These nuclei are then resuspended in cytoplasm from late gastrula stage embryos. After an incubation period, the nuclei from egg extract become smaller in late stage embryo extract. We reasoned that this would be a useful assay for identifying cytoplasmic components present in late stage embryos that contribute to developmental nuclear size scaling. Using this assay, coupled with in vivo validation, we demonstrated that protein kinase C (PKC) contributes to developmental reductions in nuclear size in X. laevis23.

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			<td>Alexa Fluor 568 Donkey anti-mouse IgG</td>
			<td>Molecular Probes</td>
			<td>A10037</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP disodium salt</td>
			<td>Sigma Aldrich</td>
			<td>A2383</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzocaine</td>
			<td>Sigma Aldrich</td>
			<td>E1501</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma Aldrich</td>
			<td>A3059</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma Aldrich</td>
			<td>C3306</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman</td>
			<td>J2-21M</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge rotor</td>
			<td>Beckman</td>
			<td>JS 13.1</td>
			<td></td>
		</tr>
		<tr>
			<td>chymostatin</td>
			<td>Sigma Aldrich</td>
			<td>C7268</td>
			<td></td>
		</tr>
		<tr>
			<td>creatine phosphate disodium</td>
			<td>Calbiochem</td>
			<td>2380</td>
			<td></td>
		</tr>
		<tr>
			<td>cycloheximide</td>
			<td>Sigma Aldrich</td>
			<td>C6255</td>
			<td></td>
		</tr>
		<tr>
			<td>cytochalasin D</td>
			<td>Sigma Aldrich</td>
			<td>C8273</td>
			<td></td>
		</tr>
		<tr>
			<td>disposable wipes (kimwipes)</td>
			<td>Sigma Aldrich</td>
			<td>Z188956</td>
			<td></td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Sigma Aldrich</td>
			<td>W326306</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma Aldrich</td>
			<td>E4378</td>
			<td></td>
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			<td>Formaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass crystallizing dish (150x75 mm)</td>
			<td>VWR</td>
			<td>89090-662</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Macron</td>
			<td>5094-16</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst - bisBenzimide H 33342 trihydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>B2261</td>
			<td></td>
		</tr>
		<tr>
			<td>HCG - Human Chorionic Gonadotropin&#160;</td>
			<td>Prospec</td>
			<td>hor-250-c</td>
			<td></td>
		</tr>
		<tr>
			<td>L15 Media</td>
			<td>Sigma Aldrich</td>
			<td>L4386</td>
			<td></td>
		</tr>
		<tr>
			<td>leupeptin</td>
			<td>Sigma Aldrich</td>
			<td>L2884</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysolecithin</td>
			<td>Sigma Aldrich</td>
			<td>L1381</td>
			<td></td>
		</tr>
		<tr>
			<td>mAb414</td>
			<td>Abcam</td>
			<td>ab24609</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>EMD</td>
			<td>MX0045-2</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma Aldrich</td>
			<td>M9397</td>
			<td></td>
		</tr>
		<tr>
			<td>Maltose</td>
			<td>Sigma Aldrich</td>
			<td>M5885</td>
			<td></td>
		</tr>
		<tr>
			<td>NP40</td>
			<td>BDH</td>
			<td>56009</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin + Streptomycin</td>
			<td>Sigma Aldrich</td>
			<td>Pp0781</td>
			<td></td>
		</tr>
		<tr>
			<td>pepstatin</td>
			<td>Sigma Aldrich</td>
			<td>P5318</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPES</td>
			<td>Sigma Aldrich</td>
			<td>P6757</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic paraffin film (parafilm)</td>
			<td>Sigma Aldrich</td>
			<td>P7793</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Mallinckrodt</td>
			<td>70100</td>
			<td></td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Baker</td>
			<td>5 3140</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSG - Pregnant Mare Serum Gonadotropin</td>
			<td>Prospec</td>
			<td>hor-272-a</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Fisher</td>
			<td>BP328</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHPO4</td>
			<td>EMD</td>
			<td>SX0720-1</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>EMD</td>
			<td>SX0590</td>
			<td></td>
		</tr>
		<tr>
			<td>Pestle</td>
			<td>Thomas Scientific</td>
			<td>3411D56</td>
			<td></td>
		</tr>
		<tr>
			<td>Round bottom glass tubes, 15 ml</td>
			<td>Corex</td>
			<td>8441</td>
			<td></td>
		</tr>
		<tr>
			<td>Secondary antibody (Alexa Fluor 568 donkey anti-mouse IgG)</td>
			<td>ThermoFisher</td>
			<td>A10037</td>
			<td></td>
		</tr>
		<tr>
			<td>sucrose</td>
			<td>Calbiochem</td>
			<td>8550</td>
			<td></td>
		</tr>
		<tr>
			<td>thermal cycler</td>
			<td>Bio-Rad</td>
			<td>T100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman</td>
			<td>L8-80M</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge rotor</td>
			<td>Beckman</td>
			<td>SW 50.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield (anti-fade mounting medium)</td>
			<td>Vector</td>
			<td>H-1000</td>
			<td></td>
		</tr>
	</tbody>
</table>

a cell-free assay using xenopus laevis embryo extracts to study mechanisms of nuclear size regulation

A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally scales with the size of the cell, notably during embryogenesis when dramatic reductions in both cell and nuclear sizes occur. Mechanisms of nuclear size regulation are largely unknown and may be relevant to cancer where altered nuclear size is a key diagnostic and prognostic parameter. In vivo approaches to identifying nuclear size regulators are complicated by the essential and complex nature of nuclear function. The in vitro approach described here to study nuclear size control takes advantage of the normal reductions in nuclear size that occur during Xenopus laevis development. First, nuclei are assembled in X. laevis egg extract. Then, these nuclei are isolated and resuspended in cytoplasm from late stage embryos. After a 30 - 90 min incubation period, nuclear surface area decreases by 20 - 60%, providing a useful assay to identify cytoplasmic components present in late stage embryos that contribute to developmental nuclear size scaling. A major advantage of this approach is the relative facility with which the egg and embryo extracts can be biochemically manipulated, allowing for the identification of novel proteins and activities that regulate nuclear size. As with any in vitro approach, validation of results in an in vivo system is important, and microinjection of X. laevis embryos is particularly appropriate for these studies.

Assembly of Nuclei in Egg Extract
The first steps of this protocol are to prepare X. laevis egg extract (Protocol 1) and demembranated sperm nuclei (Protocol 2). These reagents are then used to assemble nuclei de novo (Protocol 3). Figure 1 shows some representative data. Addition of calcium drives the meiotically arrested egg extract into interphase, and the cycloheximide keep...

Watch this Scientific Journal Video about A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation at JoVE.com

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

Mechanisms of cellular and intra-cellular scaling remain elusive. The use of Xenopus embryo extracts has become increasingly common to elucidate mechanisms of organelle size regulation. This method describes embryo extract preparation and a novel nuclear scaling assay through which mechanisms of nuclear size regulation can be identified.

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally ...

The overall goal of this procedure is to use Xenopus egg and embryo extracts to produce an assay in which large nuclei shrink smaller. This method can help answer key questions about nuclear cell biology, such as what are the mechanisms that regulate nuclear size? The main advantage of this technique is that the composition and activities of Xenopus cell-free extract are easily manipulated to assess the facts on nuclear size.After preparing Xenopus egg extracts, demembranated sperm, and assembled nuclei according to the text protocol, collect freshly laid eggs by holding frogs over a glass, reusable Petri dish. Promote egg-laying by applying gentle pressure to the lower back near the cloaca. Prop up the dish at approximately 45 degrees to allow the eggs to collect along the edge of the dish.Then, remove the excess buffer and add enough one-third X MMR to barely cover the eggs. Using a tightly-fitted pestle, macerate and homogenize 1/4 of a testis in a 1.5 milliliter tube with 500 microliters of high-salt Modified Barth's Saline, or MBS. Then add the macerated testis to the eggs and allow fertilization to occur at room temperature for 15 minutes.After the incubation, flood the eggs with one-third X MMR. Then, five to 10 minutes later, confirm fertilization by checking for contraction of the dark pigmented animal pole and by rotation of the embryos with their animal poles facing up. Using a wide bore plastic pipette, diligently remove dead, lysed, or unfertilized, uncleaved eggs as they will rapidly induce death in neighboring embryos.Anywhere from one to 2.5 hours post-fertilization, dejelly embryos in two to three percent cysteine dissolved in one-third X MMR, which has been adjusted to pH 7.9. Pryor to dejellying, embryos occupy a relatively large volume. Perform two buffer changes for three to four minutes each.Then, thoroughly wash the embryos with six to 10 brief changes of one-third X MMR to remove all traces of cysteine. After dejellying, embryos pack tightly and occupy a relatively small volume. Next, using a wide-bore glass pipette, transfer healthy, fertilized embryos to a Petri dish containing fresh one-third X MMR.And at room temperature, allow them to develop to the desired stage. Continue to remove dead or lysed embryos as indicated by a lack of first division or a white, puffy appearance. When the embryos have reached stage 11.5 to 12, transfer them into fresh one-third X MMR supplemented with 0.5 millimolar cycloheximide and incubate the embryos at room temperature for one hour to arrest them in late interphase.With a wide bore glass or plastic pipette, transfer a minimum of 15 interphase arrested embryos to a microcentrifuge tube. Add one millileter of egg lysis buffer, or ELB, supplemented with one microliter of LPC stock to the embryos. Gently invert the tube to wash the embryos.Then allow the embryos to fall to the bottom of the tube. Before removing the buffer, repeat the wash two more times. Resuspend the embryos in 500 microliters of ELB, plus 0.5 microliters of LPC stock, 5 microliters of 19.7 millimolar cycloheximide, and 5 microliters of 35.5 millimolar cytochalasin D to inhibit actin polymerization.Centrifuge the samples in a tabletop centrifuge at 200 times G for one minute, then remove the excess buffer and use a pestle to thoroughly crush the embryos. Place the crushed samples in a swinging bucket rotor and centrifuge at 10, 000 times G, and 16 degrees Celsius for 10 minutes. With a 200 microliter pipette tip, puncture the lipid layer from the top, and using a clean pipette tip, remove the middle cytoplasmic amber-colored layer to an appropriately-sized tube.Supplement the embryo extract with the following reagents. Then, gently invert the tube to mix. Then, after visualizing endogenous embryonic nuclei in the extract by preparing a squash according to the text protocol, remove the nuclei from the extract by first adding an equal volume of ELB containing the reagents listed here.Gently invert the tube to mix. Centrifuge the sample in a swinging bucket rotor at 17, 000 times G at 16 degrees Celsius for 15 minutes. Then, collect the supernatant, taking care to avoid any remaining lipid at the top, and leave the pelleted nuclei at the bottom undisturbed.Prepare a squash as described in the text protocol to ensure that most nuclei have been removed. Then use the fresh extract, or aliquot the sample, and store it at minus 80 degrees Celsius. Isolate nuclei assembled in egg extract by diluting 25 to 150 microliters of pre-assembled nuclei in one millileter of ELB in a 1.5 milliliter tube.Centrifuge at 1600 times G and four degrees Celsius for three minutes. Then remove the buffer, taking care not to disturb the pellet. Add to the pellet a volume of embryo extract equal to the original volume of egg extract, and gently tap the tube to break up the pellet and resuspend the nuclei.Incubate at room temperature for 90 minutes, gently tapping the tube to mix every 15 to 30 minutes. After the incubation, tap the tube to resuspend the nuclei just prior to adding 500 microliters of ELB with 15%glycerol and 2.6%Paraformaldehyde. Invert the tube immediately, and place the tube on a rotator at room temperature for 15 minutes.Prepare spin-down tubes by outfitting 15 milliliter, round-bottom glass tubes with round-bottom plastic inserts. Add 5 milliliters of nuclear cushion buffer, then drop a 12 millimeter circular coverslip into the tube, being sure it lays flat on top of the plastic insert. Using a wide bore pipette tip, gently layer the fixed nuclei solution on top of the nuclear cushion.Centrifuge it in a swinging bucket rotor at 1, 000 times G and 16 degrees Celsius for 15 minutes. Next, use an aspirator to remove all the buffer and hold the plastic insert to the top of the tube. Then, with a pair of fine forceps, remove the coverslip, taking care to note the side of the coverslip onto which the nuclei were spun.Host fix the coverslip in cold methanol for five minutes at room temperature. Transfer the coverslip, nuclei-side up onto a sheet of plastic paraffin film lining a large, plastic Petri dish. Then place wet, disposable wipes along the side of the dish to prevent dehydration.With 500 microliters of PBS NP40, rehydrate the nuclei on the coverslip for five to 10 seconds and aspirate. Then, carefully layer 75 microliters of PBS 3%BSA onto the coverslip, and allow it to block at room temperature for one hour, or four degrees Celsius overnight. Remove the blocking buffer and incubate with primary antibody diluted in PBS 3%BSA.Then wash the samples with five immediate changes of PBS NP40. Repeat these incubation and washing steps with secondary antibodies. Incubate with 10 micrograms per milliliter of Hoechst dilated in PBS 3%BSA for five minutes at room temperature.Then wash five times with PBS NP40 and remove all excess buffer. Finally, mount the coverslip onto a slide with five microliters of anti-fade mounting medium. Use clear nail polish to seal the coverslip and immediately image using an epifluorescence microscope or store at 40 degrees Celsius for later imaging.This figure shows the assembly of egg extract nuclei. 30-45 minutes after initiation of the reaction, the thin, S-shaped sperm nuclei should begin to thicken into slug-shaped chromatin masses. After nuclear assembly occurs, the nuclear shape should become more rounded, and the nuclear volume should increase as the nuclear envelope expands and nuclear proteins are imported.As shown here, a Hoechst stained squash of a small aliquot of the embryo extract shows many stained nuclei indicating that the preparation of extract was successful. After nuclei removal by centrifugation, compared to the first squash, very few nuclei should be left in the extract, as revealed in this panel, ensuring that endogenous nuclei do not interfere with downstream analysis. If nuclei are still present, a second round of centrifugation is required.In this nuclear shrinking assay, egg extract nuclei resuspended in late-stage embryo extract, successfully becomes smaller over time. However, nuclei incubated with heat inactivated embryo extract do not. Repeating the experiment multiple times is important to address inherent variability of the assay.Once mastered, the entire protocol can be done in two days with the nuclear shrinking assay itself taking approximately four hours. While attempting this procedure, it's important to remember to quality-check the eggs and embryos throughout and to be very careful to avoid the pellet and lipid layers when collecting cytoplasmic extract after centrifugation. Results obtained by following this procedure should be validated.For example, my embryo microinjection, or cell culture transfection. Furthermore, this method might be used to explore size regulation of other organelles. After its development, this technique paved the way for researchers in the field of nuclear cell biology to explore the regulatory mechanisms of nuclear size and the functional role of nuclear size in development and carcinogenesis.After watching this video, you should have a good understanding of how to assemble and isolate Xenopus egg extract nuclei, generate Xenopus embryos and embryo extract, perform the nuclear shrinking assay and visualize the results.

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Research

JoVE Journal

Biology

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife - a specialized dissection tool made of one's eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development.

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development of a larger region of the embryo. This makes defining its role in a particular tissue difficult to analyze. For example, noggin overexpression in Xenopus laevis embryos causes the expansion of the entire anterior region, including the eye1,2. From this result, it is not known if Noggin plays a direct role in eye determination or that by causing an expansion of neural tissue, Noggin indirectly affects eye formation. Having this complex phenotype makes studying its eye-specific role in cell fate determination difficult to analyze. We have developed an assay that overcomes this problem. Taking advantage of the pluripotent nature of the Xenopus laevis animal cap 3, we have developed an assay to test the ability of gene product(s), like noggin or the eye field transcription factors (EFTFs), to transform caps into particular tissue or cell types by transplanting this tissue onto the side of the embryo 4. While we have found either Noggin protein treatment or a collection of transcription factors can determine retinal cell fate in animal caps, this procedure could be used to identify gene product(s) involved in specifying other tissues as well.

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop.
Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group1. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle.
The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress 2. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures 3. DNA damage can be induced by ultraviolet (UV) irradiation, &gamma;-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin3, 4. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint 4-7. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint 8. The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphy's laboratory and is widely used to induce ATR/Chk1 checkpoint signaling 9-12.
Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure.

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Xenopus egg extract is a useful model system to investigate the DNA damage checkpoint. This protocol is for the preparation of Xenopus egg extracts and DNA damage checkpoint inducing reagents. These techniques are adaptable to a variety of DNA damaging approaches in the study of the DNA damage checkpoint signaling.

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...