Name: isolation of giant lampbrush chromosomes from living oocytes of frogs and salamanders
Uploaded: 2016-12-05T12:30:00
Description: Watch this Scientific Journal Video about Isolation of Giant Lampbrush Chromosomes from Living Oocytes of Frogs and Salamanders at JoVE.com

Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R01 GM33397. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. J.G.G. is American Cancer Society Professor of Developmental Genetics.

General information about frogs and salamanders, as well as sources of animals, can be found at the following websites: Xenbase (http://www.xenbase.org) and Sal-Site (http://www.ambystoma.org). This protocol follows the animal care guidelines of the Department of Embryology of the Carnegie Institution for Science.
1. Solutions
<ol>
	<li>Make 10 L &#34;frog water&#34;: Add 10 mL of 1 M CaCl2 and 10 mL of 1 M NaHCO3 to 10 L deionized or dechlorinated H2O with s...

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lampbrush+chromosomes.">Lampbrush chromosomes.</a> Xenopus laevis: Practical Uses in Cell and Molecular Biology. Vol. 36 Methods in Cell Biology , 149-166 (1991).</li><li>Gall, J. G., Wu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examining+the+contents+of+isolated+Xenopus+germinal+vesicles.">Examining the contents of isolated Xenopus germinal vesicles.</a> Methods. 51, 45-51 (2010).</li><li>Penrad-Mobayed, M., Kanhoush, R., Perrin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tips+and+tricks+for+preparing+lampbrush+chromosome+spreads+from+Xenopus+tropicalis+oocytes.">Tips and tricks for preparing lampbrush chromosome spreads from Xenopus tropicalis oocytes.</a> Methods. 51, 37-44 (2010).</li><li>Morgan, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Working+with+oocyte+nuclei:+cytological+preparations+of+active+chromatin+and+nuclear+bodies+from+amphibian+germinal+vesicles.">Working with oocyte nuclei: cytological preparations of active chromatin and nuclear bodies from amphibian germinal vesicles.</a> Methods Mol. Biol. 463, 55-66 (2008).</li><li>Paine, P. L., Johnson, M. E., Lau, Y. T., Tluczek, L. J., Miller, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+oocyte+nucleus+isolated+in+oil+retains+in+vivo+structure+and+functions.">The oocyte nucleus isolated in oil retains in vivo structure and functions.</a> Biotechniques. 13, 238-246 (1992).</li><li>Handwerger, K. E., Cordero, J. A., Gall, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cajal+bodies,+nucleoli,+and+speckles+in+the+Xenopus+oocyte+nucleus+have+a+low-density,+sponge-like+structure.">Cajal bodies, nucleoli, and speckles in the Xenopus oocyte nucleus have a low-density, sponge-like structure.</a> Mol Biol Cell. 16, 202-211 (2005).</li><li>Patel, S., Novikova, N., Beenders, B., Austin, C., Bellini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+images+of+RNA+polymerase+II+transcription+units.">Live images of RNA polymerase II transcription units.</a> Chromosome Res. 16, 223-232 (2008).</li><li>Gall, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Octagonal+nuclear+pores.">Octagonal nuclear pores.</a> J Cell Biol. 32, 391-399 (1967).</li><li>L&#246;schberger, A., 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=Super-resolution+imaging+visualizes+the+eightfold+symmetry+of+gp210+proteins+around+the+nuclear+pore+complex+and+resolves+the+central+channel+with+nanometer+resolution.">Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution.</a> J Cell Sci. 125, 570-575 (2012).</li><li>Gottfert, F., 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=Coaligned+dual-channel+STED+nanoscopy+and+molecular+diffusion+analysis+at+20+nm+resolution.">Coaligned dual-channel STED nanoscopy and molecular diffusion analysis at 20 nm resolution.</a> Biophys J. 105, L01-L03 (2013).</li><li>Wallace, R. A., Jared, D. W., Dumont, J. N., Sega, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+incorporation+by+isolated+amphibian+oocytes:+III.+Optimum+incubation+conditions.">Protein incorporation by isolated amphibian oocytes: III. Optimum incubation conditions.</a> J. Exp. Zool. 184, 321-333 (1973).</li><li>Bridger, J., Spector, D. L., Goldman, R. D., Leinwand, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situhybridization+to+DNA.">Fluorescence in situhybridization to DNA.</a> Cells: A Laboratory Manual. 3, 111.111-111.136 (1998).</li><li>Singer, R. H., Spector, D. L., Goldman, R. D., Leinwand, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+to+RNA.">In situ hybridization to RNA.</a> Cells: A Laboratory Manual. 3, 111-116 (1998).</li><li>Gardner, E. J., Nizami, Z. F., Talbot, C. C., Gall, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+intronic+sequence+RNA+(sisRNA),+a+new+class+of+noncoding+RNA+from+the+oocyte+nucleus+of+Xenopus+tropicalis.">Stable intronic sequence RNA (sisRNA), a new class of noncoding RNA from the oocyte nucleus of Xenopus tropicalis.</a> Genes Dev. 26, 2550-2559 (2012).</li><li>Talhouarne, G. J., Gall, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lariat+intronic+RNAs+in+the+cytoplasm+of+Xenopus+tropicalis+oocytes.">Lariat intronic RNAs in the cytoplasm of Xenopus tropicalis oocytes.</a> RNA. 20, 1476-1487 (2014).</li><li>Duryee, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+nuclei+and+non-mitotic+chromosome+pairs+from+frog+eggs.">Isolation of nuclei and non-mitotic chromosome pairs from frog eggs.</a> Arch. Exp. Zellforsch. 19, 171-176 (1937).</li></ol>

The first observations of &#34;living&#34; LBCs from hand-isolated GVs of frogs and salamanders were made nearly 80 years ago by the American biologist William Duryee,19 before the introduction of phase contrast and DIC microscopy, before fluorescent immunostaining, and before FISH. The advantages of LBCs for investigating details of chromosome structure and transcription at the individual gene level required development of techniques to attach the LBCs to glass slides, to fix them in a lifelike manner, and to...

Most vertebrates, with the notable exception of marsupials and placental mammals, produce large yolky eggs. Despite their sometimes enormous size, these eggs are single cells that reach their final dimension while still in the ovary of the female. Ovarian eggs are called oocytes and each typically contains a single giant nucleus, known since the early 19th century as the germinal vesicle or simply GV.1 Oocytes of the common laboratory frogs, Xenopus laevis and Xenopus tropicalis, reach a maximal diameter of 1.2 mm and 0.8 mm respectively (Figure 1). The GVs from mature oocytes of these two frogs are 0.3 - 0.4 mm in diameter (Figures 2, 3). Salamanders typically have even larger oocytes and GVs. Fully mature oocytes of the Mexican axolotl, Ambystoma mexicanum, are more than 2 mm in diameter and the GV is about 0.5 mm. Thus, these nuclei are readily visible to the naked eye and can be manipulated in many ways that are impossible with the nuclei of typical somatic cells.
Equally remarkable is the gigantic size of the chromosomes within the GV, a fact recognized already at the end of the 19th century. Individual chromosomes of Ambystoma and other salamanders can be up to 1 mm in length (Figures 4, 5). Those of Xenopus are considerable smaller, although with lengths up to 100 &#181;m or more, they dwarf the typical somatic chromosomes of most organisms. An important feature of oocyte chromosomes is their extraordinary transcriptional activity, which leads to one of their most characteristic morphological features &#8212; hundreds of paired lateral loops (Figure 5). Each loop consists of one or a few transcription units that actively synthesize RNA. The loops give oocyte chromosomes a fuzzy appearance, which led to the name &#34;lampbrush&#34; chromosome after their superficial resemblance to the brushes used in earlier times to clean kerosene lamp chimneys.2
The focus of this paper is on the use of isolated GVs to study LBCs and nuclear organelles (nucleoli, histone locus bodies, and speckles). Two rather different techniques will be described. In the first, more common technique, GVs are isolated in a saline solution using jeweler&#39;s forceps, briefly rinsed to remove adherent yolk, and the nuclear envelope is removed, again with jeweler&#39;s forceps. The gelatinous contents, containing the LBCs and nuclear organelles, are allowed to settle onto a glass microscope slide or coverslip. Such preparations can be examined directly by phase contrast or DIC microscopy. Alternatively, preparations may be centrifuged to attach the LBCs and organelles to the slide or coverslip. Such preparations can then be processed for detailed molecular analysis of nucleic acids and proteins, primarily by immunofluorescence and fluorescent in situ hybridization (FISH).3-7
A second technique involves isolation of the GV in mineral oil.8 Oil-isolated GVs remain transcriptionally active for many hours and are potentially useful for studies where one wants the nuclear contents to be as lifelike as possible.9,10 Because the refractive index of the nuclear &#34;sap&#34; is close to that of the LBCs and other nuclear organelles (Figure 3), microscopical techniques can be a challenge with oil-isolated GVs.
Finally, because of their size and ease of manipulation, GVs are ideal material for studies on the nuclear envelope. The nuclear pore complex was first described from electron microscopic studies on amphibian GV envelopes11 and more recent superresolution observations have used the same material.12,13

<table border="0" cellpadding="0" cellspacing="0" width="1562">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Paraformaldehyde (reagent grade, crystalline)</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:213px;">P6148-500G</td>
			<td style="width:643px;">Despite warnings in many protocols, a concentrated solution can be stored indefinitely at room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethyl 3-aminobenzoate methanesulfonate</td>
			<td>Sigma-Aldrich</td>
			<td>A5040-100G</td>
			<td>Sometimes referred to as MS-222</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethicon RB-1 1/2 circle taper point 3-0 sutures</td>
			<td>VWR&#160;</td>
			<td>95057-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraplast (paraffin wax)</td>
			<td>Sigma-Aldrich</td>
			<td>P3558-1KG</td>
			<td style="width:643px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">p-Phenylenediamine</td>
			<td>Sigma-Aldrich</td>
			<td>P6001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin</td>
			<td>Grocery Store</td>
			<td></td>
			<td>Commercial Knox gelatin works fine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProLong Gold antifade mountant</td>
			<td>ThermoFisher Scientific</td>
			<td>P10144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gold Seal cover glass&#160; 22 x 22 mm #1 1/2 (0.16-0.19 mm thick)</td>
			<td>Electron Microscopy Sciences</td>
			<td>63786-01</td>
			<td>These coverslips are the recommended thickness for superresolution microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont forceps #5</td>
			<td>Electron Microscopy Sciences</td>
			<td>72700-D</td>
			<td>http://www.emsdiasum.com/microscopy/products/tweezers/dumont_positive_action.aspx</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraffin oil (light)&#160;</td>
			<td>EMD Chemicals</td>
			<td>PX0047-1</td>
			<td>For isolating GVs in oil</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adhesive in situ PCR and hybridization chambers (25 &#181;l).</td>
			<td>BioRad</td>
			<td>Frame-Seal Slide Chambers #SLF0201</td>
			<td>http://www.bio-rad.com/en-us/sku/slf0201-frame-seal-slide-chambers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicone isolators</td>
			<td>Grace-Biolabs</td>
			<td>select from catalog link</td>
			<td>http://www.gracebio.com/life-science-products/microfluidics/silicone-isolators.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coplin jars and staining dishes</td>
			<td>Electron Microscopy Sciences</td>
			<td>select from catalog link</td>
			<td>http://www.emsdiasum.com/microscopy/products/histology/staining.aspx</td>
		</tr>
	</tbody>
</table>

isolation of giant lampbrush chromosomes from living oocytes of frogs and salamanders

We describe methods for studying the giant transcriptionally active lampbrush chromosomes (LBCs) found in the oocyte, or unlaid egg, of frogs and salamanders. Individual LBCs can be up to 1 mm in length and they reside in a gigantic nucleus, itself up to 0.5 mm in diameter. The large size of the chromosomes permits unparalleled observations of active genes by light optical microscopy, but at the same time special techniques are required for isolating the nucleus, removing the nuclear envelope, and spreading the chromosomes on a microscope slide. The oocyte nucleus, also called the germinal vesicle (GV), is isolated in a medium that allows partial gelling of the nuclear actin and preserves the delicate structure of the LBCs. This step is carried out manually under a dissecting microscope using jeweler&#39;s forceps. Next, the nuclear envelope is removed, again manually with jeweler&#39;s forceps. The nuclear contents are quickly transferred to a medium that disperses the actin gel and allows the undamaged LBCs to settle onto a microscope slide. At this point the LBCs and other nuclear organelles can be viewed by phase contrast or differential interference contrast microscopy, although finer details are obscured by Brownian motion. For high resolution microscopical observation or molecular analysis, the whole preparation is centrifuged to attach the delicate LBCs firmly to the slide. A brief fixation in paraformaldehyde is then followed by immunofluorescent staining or in situ hybridization. LBCs are in a transcriptionally active state and their enormous size permits molecular analysis at the individual gene level using confocal or super-resolution microscopy.

To examine giant lampbrush chromosomes one begins by isolating oocytes from a frog or salamander. Figure 1 shows a group of mature oocytes in a buffered saline solution after removal from the ovary of the frog, Xenopus. Such oocytes remain in good condition for days at room temperature. The nucleus (or germinal vesicle) is then removed from an oocyte with jeweler&#39;s forceps, either in a saline solution (Figure 2) or in oil (Figure 3</s...

Watch this Scientific Journal Video about Isolation of Giant Lampbrush Chromosomes from Living Oocytes of Frogs and Salamanders at JoVE.com

Isolation of Giant Lampbrush Chromosomes from Living Oocytes of Frogs and Salamanders

We present simple techniques for isolating giant transcriptionally active lampbrush chromosomes from living oocytes of frogs and salamanders. We describe how to observe these chromosomes &#34;alive&#34; by phase contrast or differential interference contrast, and how to fix them for fluorescent in situ hybridization or immunofluorescent staining.

We describe methods for studying the giant transcriptionally active lampbrush chromosomes (LBCs) found in the oocyte, or unlaid egg, of frogs and ...

The overall goal of this procedure is to isolate the giant nucleus, or germinal vesicle, from an amphibian oocyte, and to spread the lampbrush chromosomes, and other nuclear organelles, on a microscope slide for immunostaining, in citu hybridization, and other molecular techniques. This method can help answer key questions about the basic structure of chromosomes, nucleoli, and other nuclear organelles as well as molecular details of transcription, splicing, and RNA export. The main advantage of this technique is the ease with which chromosome structure and transcription can be examined by a conventional light microscopy, using immunofluorescence, in citu hybridization, and other molecular techniques.Demonstrating the procedure will be Dr.Zehra Nizami, a postdoc from my laboratory. After preparing all of the required solutions, assemble well slides, composed of subbed, glass slides, to which plastic wells have been attached with paraffin, as discussed in the text protocol. Then gather ovarian tissue previously collected from an adult, female frog or a salamander, and fill a small Petri dish with oocyte culture medium.Proceed to place a portion of the isolated tissue, one containing approximately 10 to 20 oocytes, into this plate. Next, identify a single oocyte and use two pairs of jewelers forceps to sever the stalk that connects it to the ovary wall. Once it is freed, transfer the oocyte to a new Petri dish containing germinal vesicle, or GV, isolation medium.Under low magnification, locate the animal pole of the oocyte, which can be identified by its dark coloration. Then use forceps to poke a large hole near the animal pole and proceed to gently squeeze out the transparent oocyte nucleus, also referred to as the GV.Afterwards, roll the GV away from any yolk that was also extruded. To remove yolk that remains tightly adhered, draw the nucleus in and out of a pipette with a tip having a diameter slightly larger than that of the GV.Following the collection of a frog GV, immediately transfer it to dispersal medium in order to prevent nuclear hardening that would interfere with subsequent steps.Next, with two pairs of forceps, grasp the nuclear envelope near the top of the GV, taking care not to press down. While still gripping the envelope, proceed to pull the two forceps apart and expect the contents of the nucleus to pop out. To collect these contents, first prepare an adjustable 20 microliter pipette with a cut tip as described in the text protocol.Then, from the dish, proceed to draw up five microliters of dispersal liquid followed by five microliters of the solution with the GV contents. Once these contents have been collected, transfer them to a well slide filled with dispersal solution. Ensure that the liquid in the slide has a convex surface so as to avoid the trapping of bubbles in subsequent steps.Afterwards, position an 18 millimeter cover slip over the well and place the assembled slide into a moist chamber for 10 to 60 minutes. During this time, expect the nuclear gel to slowly disperse, so that the chromosomes and nuclear organelles come to lie on the surface of the glass. After removing a nucleus from a salamander oocyte, leave it in the isolation medium.Note that salamander GVs, unlike those of the frog species used here, will not unduly harden in this solution. Remove the nuclear envelope from the salamander GV using the same forceps-based technique as described for frogs. Next, pick up the slightly gelled nuclear contents with a pipette having a cut tip, and transfer the gel to a Petri dish containing dispersal solution.Quickly rinse the pipette in the dispersal solution, and use it to collect the nuclear gel. Then introduce the gel into a well slide, add a cover slip, and allow the gel to disperse as previously described. Then use phase-contrast to view the nuclear contents, and identify the lampbrush chromosomes, abbreviated as LBCs, by their large size and paired, lateral loops.After confirming that the LBCs are unbroken, prepare the slide for centrifugation by placing a piece of filter paper on the cover slip. Then press down to remove excess liquid from the chamber. Next, add petroleum jelly to each edge of the cover slip.Heat a metal rod to approximately 70 to 80 degrees Celsius and use it to melt the jelly and seal the cover slip onto the underlying plastic. Repeat this procedure for other slides with LBCs of good quality. Once all the cover slips have been affixed, position the slides in carriers for centrifugation and ensure that opposite carriers are balanced.Using the slow start function, centrifuge the slides as described in the text using custom-built carriers. Alternatively, a standard tabletop centrifuge can be used where slides are taped to a plate and spun. To prepare the nuclear contents for immunostaining or in citu hybridization, first wipe away the temporary Vaseline seal with a razor blade, and then place the slide into the rack of a standard slide-staining dish.Next, add enough paraformaldehyde to cover the slides. Upon submergence, use blunt forceps to remove and discard each cover slip. Leave the slides in fixative for 15 to 30 minutes.Following fixation, place a slide into a staining dish containing ice cold PBS. Then insert a sharp razor blade at the edge of the plastic square, and pry this plastic well loose. Once this process has been repeated for all the slides, proceed with staining as discussed in the text protocol.Shown in this dark-field illumination image are the nuclear contents from a single axolotl GV in prophase of the first meiotic division, collected and prepared using the described protocol. 14 large, paired LBCs are apparent, as are amplified nucleoli. Additional staining of this preparation, with an antibody against phosporylted RNA polymerase II, shown here in green, reveals the locations of histone locus bodies and also provides insight into the robust, transcriptional activity of these oocyte chromosomes.Higher magnification of a single axolotl LBC, in this instance one that has been stained green for RNA polymerase and blue for DNA, demonstrates the most unique morphological feature of these chromosomes. Hundreds of paired lateral loops, each of which represents at least one transcription unit that actively synthesizes RNA. Comparison with a similarly stained frog LBC, imaged at the same magnification, emphasizes the enormous size difference between frog and salamander chromosomes, a difference that correlates with the total DNA content of the genomes of the species used.However, even in the frog chromosome, the individual transcription units can be seen as loops of chromatin projecting laterally from the chromosome axis, as shown in this higher magnification image. Oocytes of fish, birds, reptiles, and some invertebrates also contain giant lampbrush chromosomes. Remarkably, lampbrush chromosomes are generated when sperm heads from other organisms, including humans, are injected into the amphibian germinal vesicle.Once the technique is mastered, a batch of AGV spreads can be prepared in one hour, and then centrifuged and fixed within another one to two hours. After watching this video, you should have a good understanding of how to isolate a GV and spread the lampbrush chromosomes for further study using molecular probes.

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