Name: isolation of giant lampbrush chromosomes from living oocytes of frogs and salamanders
Uploaded: 2016-12-05T12:30:00.000+00:00
Duration: 10 min 7 s
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 stirring.</li>
	<li>Make 1 L 20% paraformaldehyde: Make 1 L of 4 mM Na2CO3. In a chemical hood add 200 g of powdered paraformaldehyde, and heat to about 80 oC to dissolve. Cool, and filter through filter paper. Caution: paraformaldehyde is toxic and must be handled with care. Alternatively, purchase commercial paraformaldehyde solution.</li>
	<li>Make 1 L amphibian anesthetic solution: Add 1.5 g of ethyl 3-aminobenzoate methanesulfonate to 1 L frog water. Store at room temperature.</li>
	<li>Make 1 L oocyte culture medium OR214: 82.5 mM NaCl, 2.5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM Na2HPO4, 5 mM HEPES (from 500 mM stock, pH 8.3). The final pH will be about pH 7.8. Add 100 mg ampicillin and 100 mg streptomycin to prevent bacterial growth.</li>
	<li>Make 1 L GV isolation solution: 83 mM KCl, 17 mM NaCl, 6.5 mM Na2HPO4, 3.5 mM KH2PO4, 1 mM MgCl2, 1 mM dithiothreitol (DTT). Adjust to pH 7.0.</li>
	<li>Make 100 mL GV dispersal solution: 20.7 mM KCl, 4.3 mM NaCl, 1.6 mM Na2HPO4, 0.9 mM KH2PO4, 1 mM MgCl2, 1 mM dithiothreitol (DTT), 0.1% paraformaldehyde. Adjust to pH 7.0. To facilitate more rapid dispersal of the nuclear gel, add 10-50 &#181;M CaCl2.</li>
	<li>Make 500 mL subbing solution. Swell 0.5 g of commercial gelatin in 20 mL H2O for 5 - 10 min. Add 500 mL boiling H2O and carefully swirl to dissolve the gelatin. Cool and add 50 mg CrK(SO4)2 &#183;12 H2O. Filter with suction and store at 4 oC.</li>
	<li>Make 10 mL mounting medium. Dissolve 10 mg phenylenediamine in 5 mL PBS (135 mM NaCl, 2.5 mM KCl, 4.3 mM Na2HPO4, 1.5 mM KH2PO4, adjusted to pH 9). Add 5 mL glycerol. Store in 250 &#181;L aliquots at -80 oC.</li>
</ol>
2. Materials
<ol>
	<li>For subbed slides, place 3 inch x 1 inch glass microscope slides in a commercial detergent solution and rub them to remove any contaminants. Place in a slide rack and rinse well in tap water, then in deionized water. Dip the slides in the subbing solution, drain at an angle, and bake overnight at 60 oC.</li>
	<li>For plastic squares, cut 25 mm squares from a sheet of 1 mm thick poly(methyl methacrylate). Drill a 5 mm round hole in the center of each square. Sand both faces and the edges of the square as well as the circumference of the hole. 
	NOTE: As an alternative to homemade plastic squares and well slides, as described in the next paragraph, use commercial adhesive in situ PCR and hybridization chambers (25 &#181;L). One could probably also use re-usable silicone isolators for cell culture, although this has not been thoroughly tested as yet.</li>
	<li>For well slides, melt 20 - 30 g of paraffin wax (56 - 57 oC) in a 50 mL glass beaker on a hot plate. With a 20 &#181;L pipette, spread one 5 &#181;L drop of paraffin on each side of the center of a subbed slide. Press a plastic square into the paraffin and place the slide onto the hot plate. The paraffin will melt and should spread evenly under the plastic square.
	<ol>
		<li>Remove the slide from the hot plate and let it cool. Place a support under each end of the slide, so that the slide does not contact the tabletop during the cooling process. If the paraffin cools too rapidly, it may pull away from the central hole. Prepare 10 or more well slides: each GV preparation uses a new well slide.</li>
	</ol>
	</li>
	<li>For slide carriers for centrifugation, use specially constructed carriers for the rotor used. Several manufacturers provide centrifuges with carriers for 96 - well plastic plates. These carriers can be adapted quite easily to hold microscope slides.</li>
</ol>
3. Isolation of Oocytes
<ol>
	<li>Place an adult female frog (Xenopus laevis or Xenopus tropicalis) or salamander (Ambystoma mexicanum) in enough anesthetic solution to cover the animal completely. When the animal ceases movement, place it belly-up on a bed of chipped ice in a suitable container.</li>
	<li>With surgical scissors make a 2 - 3 cm incision in the lower abdomen lateral to the midline. Move the internal organs gently aside to find the ovary on the side of the incision (there are two ovaries, one on each side of the abdomen). Cut out a portion of the ovary with enough oocytes for the planned experiments. Since there are thousands of oocytes in each ovary, a relatively small piece of ovary (1 - 3 cm) will usually suffice.</li>
	<li>Place one or a few pieces of ovary in OR2 saline in a large petri dish (100 mm) at room temperature. Oocytes can be stored in good condition for several days, if damaged oocytes are removed and the OR2 solution is changed daily.</li>
	<li>Suture the incision with surgical silk and return the animal to an individual glass bowl for recovery. Add just enough &#34;frog water&#34; to cover the animal until it regains consciousness (about 15 min). After the animal shows voluntary movements, fill the bowl with water. Although fresh water should be used, sterility is not necessary, as amphibians almost never become infected after surgery. If desired, neomycin can be applied to the sutures. The wound usually heals completely after a few days and the same animal can be used again after about 2 months.</li>
</ol>
4. Isolation of a Germinal Vesicle (GV)
<ol>
	<li>Place a portion of ovary (10 - 20 large oocytes) in a small petri dish (35 x 10 mm) containing OR2 solution.</li>
	<li>Remove an oocyte from the ovary using two pairs of #5 jeweler&#39;s forceps to tear the thin stalk of tissue that connects the oocyte to the ovary wall. Place the oocyte in another small petri dish containing GV isolation medium. 
	NOTE: The state of the LBCs depends on the size (maturity) of the oocyte. LBCs reach maximal length with prominent loops in oocytes that are slightly less than full size. The most mature oocytes often contain short chromosomes with contracted loops.</li>
	<li>Perform the following steps under low magnification of a dissecting microscope (field diameter approximately 10 - 20 mm).
	<ol>
		<li>Grasp the oocyte with both pairs of forceps near the animal pole (dark hemisphere) and make a small tear. Look in the extruded yolk for the transparent oocyte nucleus, also called the germinal vesicle or GV.</li>
		<li>Alternatively, poke a large hole near the animal pole with one pair of forceps and gently squeeze out the GV along with yolk (Figure 2).</li>
		<li>Gently roll the GV away from the bulk of the yolk. Usually there will be a small amount of yolk tightly adherent to the GV.</li>
	</ol>
	</li>
</ol>
5. Removal of the Nuclear Envelope
<ol>
	<li>For X. laevis and X. tropicalis, transfer the GV from the isolation medium to the spreading medium within 60 s of isolation, even if there is still a little adherent yolk. 
	NOTE: GVs of these two species &#34;harden&#34; within about 60 s if left in the isolation medium and will not spread in subsequent steps. Therefore speed is of the utmost importance when examining Xenopus GV contents.
	<ol>
		<li>Grasp the nuclear envelope near the top of the GV with one pair of jeweler&#39;s forceps, taking care not to press down. Grasp the envelope with a second pair of forceps as close to the first as possible. Pull the two pairs of forceps away from each other and the GV contents will &#34;pop&#34; out free of the envelope, which adheres tightly to one or both forceps.</li>
		<li>Pick up the GV contents in a glass pipette or an adjustable 20 &#181;L micropipette. If using an adjustable pipette, set the pipette to 10 &#181;L and cut off the end of the plastic tip with a razor blade. Draw in 5 &#181;L of liquid before picking up the GV in the remaining 5 &#181;L.</li>
		<li>Transfer the still-gelled nuclear contents to the center of a well slide previously filled with spreading solution. The spreading solution must have a convex surface so that a bubble is not trapped when the coverslip is added.</li>
		<li>Add a coverslip (18 mm) and place the slide in a moist chamber. The nuclear gel will slowly disperse over the next 10 - 60 min, so that the chromosomes and nuclear organelles come to lie on the surface of the glass slide. 
		NOTE: GVs of the salamanders A. mexicanum and N. viridescens do not &#34;harden&#34; unduly in the isolation medium. Therefore one can proceed more leisurely with removal of the nuclear envelope than one can with Xenopus.</li>
	</ol>
	</li>
	<li>Remove the nuclear envelope from a salamander GV as described in section 5.1.1, but do so in the isolation medium.
	<ol>
		<li>Pick up the slightly gelled GV contents with a pipette and transfer to a petri dish containing spreading solution.</li>
		<li>Quickly rinse out the pipette tip in the spreading solution, pick up the nuclear gel, and place it in the center of a well slide previously filled with spreading solution. Add an 18 mm coverslip and place the entire slide in a moist chamber. The nuclear sap will slowly disperse over the next 10 - 60 min and the chromosomes and nuclear organelles will come to lie on the glass surface.</li>
	</ol>
	</li>
</ol>
6. Preliminary Observation of LBCs and Organelles
<ol>
	<li>View the LBCs and nuclear organelles by phase contrast (conventional upright microscope) at low magnification after dispersal of the nuclear contents in the spreading chamber. Because the chamber is approximately 1 mm deep and the nuclear contents are at the bottom, use a low magnification objective with a long working distance (5X - 10X). 
	NOTE: The major reason for examining the preparation at this time is to determine whether it is worth carrying through the next steps. In a good preparation the chromosomes are unbroken and have settled to the bottom of the chamber. Proceed to the centrifugation step within an hour of making the preparation, as this will ensure optimal attachment of the lampbrush chromosome loops to the slide.</li>
</ol>
7. Centrifugation of the Nuclear Contents
<ol>
	<li>Place a piece of filter paper on the coverslip and press down to remove excess liquid from the chamber.</li>
	<li>Put about 1 mm3 of petroleum jelly on each side of the coverslip. Melt the petroleum jelly with a metal rod heated to about 70 - 80 oC to seal the coverslip onto the underlying plastic square.</li>
	<li>Place slides in the slide carriers for centrifugation, making sure that opposite carriers are balanced. Centrifuge the slides at about 4,800 x g for 30 min - 1 h. at 4 oC. Use the slow start feature on the centrifuge.</li>
</ol>
8. Fixing the Nuclear Contents
<ol>
	<li>For most subsequent procedures, fix the nuclear contents with paraformaldehyde.</li>
	<li>Put a slide rack into a standard microscope slide staining dish and then place the slides individually in the rack. Add enough fixative to cover the slides(2 - 4% paraformaldehyde in OR2 or PBS).</li>
	<li>With a pair of blunt forceps, push the coverslip laterally, pick it up, and discard it in the appropriate glass trash. Leave slides in the fixative for at least 15 - 30 min. For in situ hybridization and most antibody stains, longer periods of fixation (h or d) are permissible.</li>
	<li>To remove the plastic square, place slides one at a time into ice cold OR2 or PBS in a staining dish. Insert a sharp razor blade at the edge of the plastic square and pry it loose. 
	NOTE: Ideally the plastic will come off cleanly, leaving all the paraffin wax on the slide. The wax serves a useful purpose: since it surrounds the small central area where the nuclear contents are attached to the slide, it acts as a dam that allows one to work with small volumes (5 - 10 &#181;L) of reagents for immunostaining, etc. Slides can be held indefinitely in cold OR2 or PBS.</li>
</ol>
9. Immunostaining of LBCs and Nuclear Organelles
<ol>
	<li>Remove a fixed GV preparation from storage in OR2 or PBS. Wipe off excess liquid and place on supports in a moist chamber. A convenient chamber is a 90 x 15 mm petri dish containing a 90 mm circle of moist filter paper. 
	NOTE: At this point the nuclear contents are firmly attached to the slide in a 5 mm circular area free of paraffin wax. Because the wax is water-repellent, one can change solutions by adding and removing small volumes of liquid to the edge of the central area with a 20 &#181;L pipette. The major precaution is to avoid touching the wax-free area with the pipette tip.</li>
	<li>Carry out immunostaining with small volumes (10 - 20 &#181;L) of reagents. Because the chromosomes and nuclear organelles are very small and in immediate contact with added reagents, staining protocols can be short. Primary and secondary antibody staining times can be as short as 1 hr or less. Do not include detergent in any step, as this causes damage to the preparation. If desired, stain the preparation with DAPI (1 &#181;g/mL) for 10 - 20 min to accentuate the axes of the LBCs.</li>
	<li>Mount in glycerol or a commercial mounting medium suitable for immunofluorescence. To avoid trapping an air bubble during the mounting step, proceed as follows.
	<ol>
		<li>Pull 15 &#181;L of mounting medium into the tip of a 20 &#181;L pipette. Remove as much liquid as possible from the area immediately around the nuclear spread by &#34;wicking&#34; it off with a piece of filter paper (cut in the shape of a very thin triangle from a 90 mm circle of #1 filter paper).</li>
		<li>Pipette most of the mounting medium onto the preparation, being careful not to touch the area of the nuclear spread. Place the last 1 &#181;L or so of mounting medium in the center of a 22 mm coverslip (thickness #1.5). Invert the coverslip over the preparation and drop carefully into place.</li>
		<li>For temporary mounts, up to a few days, use 1 mg/mL phenylenediamine in 50% glycerol. For permanent mounts, especially for superresolution studies, use a low-fluorescence medium that hardens to a refractive index of about 1.46.</li>
	</ol>
	</li>
</ol>
10. Fluorescent In Situ Hybridization (FISH) of LBCs and Nuclear Organelles
<ol>
	<li>Because FISH protocols usually require a step at elevated temperature, remove the paraffin wax from the preparation. Carefully scrape with a razor blade, although the procedure is tedious and can lead to accidental damage of the preparation. It is useful to mark the area of the preparation on the back of the slide with a diamond point.
	<ol>
		<li>Alternatively, dissolve the paraffin wax with xylene. Set up a series of Coplin jars or staining dishes containing: 30%, 50%, 70%, 95%, and 100% ethanol; 3 containers of xylene; 2 more containers of 100% ethanol and one acetone.</li>
		<li>Place slides in the slide carrier and dehydrate them in the ethanol series, leaving slides 3 - 5 min in each concentration (shorter times if agitated).</li>
		<li>Dissolve the paraffin wax by passing the slides through the 3 containers of xylene, about 5 min each if agitated. Remove the xylene by passing through two 100% ethanols, and finally remove the ethanol with acetone. Take the slides from acetone and tilt them to dry.</li>
	</ol>
	</li>
	<li>Carry out in situ hybridization using any standard protocol.15,16</li>
</ol>
11. Isolation of a GV under Oil
<ol>
	<li>Pick up an oocyte from the OR2 solution with jeweler&#39;s forceps, including as little surrounding tissue as possible. Place the oocyte on a small piece of #1 filter paper. Excess OR2 solution will transfer to the filter paper, leaving the oocyte almost free of liquid. Pick up the &#34;dry&#34; oocyte and place it under the surface of light weight mineral oil (paraffin oil) in a small petri dish (35 x 10 mm).
	<ol>
		<li>With a fine tungsten needle or one tine of the forceps, poke a small hole in the oocyte near the dark animal pole. Pick up the oocyte with one pair of forceps (avoiding the tips) and squeeze gently. The GV will extrude along with a smaller or larger amount of yolk on its surface (Figure 3).</li>
		<li>Remove as much yolk as possible by gently sweeping the GV with the side of the forceps. In most cases it will be difficult to remove all of the yolk. However, for most purposes a small amount of yolk is not a problem.</li>
	</ol>
	</li>
	<li>To observe the GV contents, flatten the GV to a greater or lesser extent.
	<ol>
		<li>Remove the plastic square from a well slide and use the remaining paraffin wax as a shallow receptacle for paraffin oil.10 Pick up the GV along with paraffin oil in a 20 &#181;L pipette and transfer to the center of the paraffin area. Add a coverslip and observe the contents of the partially flattened GV. This technique does not damage the LBCs.</li>
	</ol>
	</li>
	<li>Alternatively, flatten the GV to approximately 10 &#181;m thickness for observation of nuclear organelles.
	<ol>
		<li>Cut off the final 1 mm of a plastic pipette tip with a sharp razor blade. Suck the GV into the tip along with exactly 5 &#181;L of mineral oil. Extrude the GV and all the oil onto the center of a 22 mm glass coverslip.</li>
		<li>Lower a standard 3&#34; x 1&#34; glass slide slowly until it just touches the oil drop. The drop and coverslip will be lifted by capillarity and the oil will spread to the edges of the coverslip. When the drop has spread completely, the oil will be approximately 10 &#181;m thick. The LBCs will be damaged and large nucleoli will be compressed slightly, but most of the smaller nuclear organelles will appear more or less as they exist in the intact GV (Figure 3).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Purkinje, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Symbolae ad ovi avium historiam ante incubationem. , (1830).</li><li>R&#252;ckert, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zur+Entwickelungsgeschichte+des+Ovarialeies+bei+Selachiern.">Zur Entwickelungsgeschichte des Ovarialeies bei Selachiern.</a> Anat. Anz. 7, 107-158 (1892).</li><li>Callan, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lampbrush Chromosomes. 36, (1986).</li><li>Gall, J. G., Callan, H. G., Wu, Z., Murphy, C., Kay, B. K., Peng, H. 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. 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The authors have nothing to disclose.

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><tbody><tr><td>Paraformaldehyde (reagent grade, crystalline)</td><td>Sigma-Aldrich</td><td>P6148-500G</td><td>Despite warnings in many protocols, a concentrated solution can be stored indefinitely at RT</td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate</td><td>Sigma-Aldrich</td><td>A5040-100G</td><td>Sometimes referred to as MS-222</td></tr><tr><td>Ethicon RB-1 1/2 circle taper point 3-0 sutures</td><td>VWR&amp;nbsp;</td><td>95057-000</td><td></td></tr><tr><td>Paraplast (paraffin wax)</td><td>Sigma-Aldrich</td><td>P3558-1KG</td><td></td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-Phenylenediamine</td><td>Sigma-Aldrich</td><td>P6001</td><td></td></tr><tr><td>Gelatin</td><td>Grocery Store</td><td></td><td>Commercial Knox gelatin works fine</td></tr><tr><td>ProLong Gold antifade mountant</td><td>ThermoFisher Scientific</td><td>P10144</td><td></td></tr><tr><td>Gold Seal cover glass&amp;nbsp; 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><td>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><td>Paraffin oil (light)&amp;nbsp;</td><td>EMD Chemicals</td><td>PX0047-1</td><td>For isolating GVs in oil</td></tr><tr><td>Adhesive &lt;em&gt;in situ&lt;/em&gt; PCR and hybridization chambers (25 &amp;micro;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><td>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><td>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...

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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 ...

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12.9K Views.  Carnegie Institution for Science. 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 "alive" by phase contrast or differential interference contrast, and how to fix them for fluorescent in situ hybridization or immunofluorescent staining.

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

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Developmental Biology

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts prepared from Xenopus laevis eggs have been powerful in dissecting biochemical networks underlying the cell-cycle progression, their ensemble average measurement typically leads to a damped oscillation, despite each individual oscillator being sustained. This is due to the difficulty of perfect synchronization among individual oscillators in noisy biological systems. To retrieve the single-cell dynamics of the oscillator, we developed a droplet-based artificial cell system that can reconstitute mitotic cycles in cell-like compartments encapsulating cycling cytoplasmic extracts of Xenopus laevis eggs. These simple cytoplasmic-only cells exhibit sustained oscillations for over 30 cycles. To build more complicated cells with nuclei, we added demembranated sperm chromatin to trigger nuclei self-assembly in the system. We observed a periodic progression of chromosome condensation/decondensation and nuclei envelop breakdown/reformation, like in real cells. This indicates that the mitotic oscillator functions faithfully to drive multiple downstream mitotic events. We simultaneously tracked the dynamics of the mitotic oscillator and downstream processes in individual droplets using multi-channel time-lapse fluorescence microscopy. The artificial cell-cycle system provides a high-throughput framework for quantitative manipulation and analysis of mitotic oscillations with single-cell resolution, which likely provides important insights into the regulatory machinery and functions of the clock.

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

We present a method for the generation of in vitro self-sustained mitotic oscillations at the single-cell level by encapsulating egg extracts of Xenopus laevis in water-in-oil microemulsions.

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts ...

Biochemistry

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2-4. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5-8. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9-12, molecular mechanisms of neurotransmitter release13-15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18-19.

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

Neuroscience

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

Biology

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Analysis of Transforming Growth Factor &#223; Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos

The two arms of the Transforming Growth Factor &#223; (Tgf&#223;) superfamily, represented by Tgf&#223;/Nodal or Bone morphogenetic protein (Bmp) ligands, respectively, play essential roles in embryonic development and adult homeostasis. Members of the Tgf&#223; family are made as inactive precursors that dimerize and fold within the endoplasmic reticulum. The precursor is subsequently cleaved into ligand and prodomain fragments. Although only the dimeric ligand can engage Tgf&#223; receptors and activate downstream signaling, there is growing recognition that the prodomain moiety contributes to ligand activity. This article describes a protocol that can be used to identify cleavage products generated during activation of Tgf&#223; precursor proteins. RNA encoding Tgf&#223; precursors are first microinjected into X. laevis embryos. The following day, cleavage products are collected from the blastocoele of&#160;gastrula stage embryos and analyzed on Western blots. This protocol can be completed relatively quickly, does not require expensive reagents and provides a source of concentrated Tgf&#223; cleavage products under physiologic conditions.

Analysis of Transforming Growth Factor &#223; Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos

When Transforming Growth Factor &#223; family precursor proteins are ectopically expressed in Xenopus laevis embryos, they dimerize, get cleaved and are secreted into the blastocoele, which begins at the late blastula to early gastrula stage. We describe a method for aspirating cleavage products from the blastocoele cavity for immunoblot analysis.

The two arms of the Transforming Growth Factor &#223; (Tgf&#223;) superfamily, represented by Tgf&#223;/Nodal or Bone morphogenetic protein (Bmp) ligands, ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

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 ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...

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

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Chapters in this video

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Fertilization of Xenopus oocytes using the Host Transfer Method

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Analysis of Transforming Growth Factor ß Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

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

Xenopus laevis as a Model to Identify Translation Impairment

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis