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. 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. 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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 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 ...

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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

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly defined states of chromatin organization. The dynamic architecture of the nucleus is controlled by multiple mechanisms and shapes the transcriptional output programs. It is, therefore, important to determine locus-specific chromatin accessibility in a reliable fashion that is preferably independent from antibodies, which can be a potentially confounding source of experimental variability. Chromatin accessibility can be measured by various methods, including the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay, that allow the determination of general chromatin accessibility in a relatively low number of cells. Here we describe a FAIRE protocol that allows simple, reliable, and fast identification of genomic regions with a low protein occupancy. In this method, the DNA is covalently bound to the chromatin proteins using formaldehyde as a crosslinking agent and sheared to small pieces. The free DNA is afterwards enriched using phenol:chloroform extraction. The ratio of free DNA is determined by quantitative polymerase chain reaction (qPCR) or DNA sequencing (DNA-seq) compared to a control sample representing total DNA. The regions with a looser chromatin structure are enriched in the free DNA sample, thus allowing the identification of genomic regions with lower chromatin compaction.

The plasticity of nuclear architecture is controlled by dynamic epigenetic mechanisms including non-coding RNAs, DNA methylation, nucleosome repositioning as well as histone composition and modification. Here we describe a Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) protocol which allows the determination of chromatin accessibility in a reproducible, inexpensive, and straightforward manner.

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies.
The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell products.

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. Recent findings revealed that TERRA molecules also interact with internal chromosomal regions to regulate gene expression in mouse embryonic stem (ES) cells. In line with this evidence, RNA fluorescence in situ hybridization (RNA-FISH) analyses have shown that only a subset of TERRA transcripts localize at chromosome ends. A better understanding of the dynamics of TERRA molecules will help define their function and mechanisms of action. Here, we describe a method to label and visualize single-telomere TERRA transcripts in cancer cells using the MS2-GFP system. To this aim, we present a protocol to generate stable clones, using the AGS human stomach cancer cell line, containing MS2 sequences integrated at a single subtelomere. Transcription of TERRA from the MS2-tagged telomere results in the expression of MS2-tagged TERRA molecules that are visualized by live-cell fluorescence microscopy upon co-expression of a MS2 RNA-binding protein fused to GFP (MS2-GFP). This approach enables researchers to study the dynamics of single-telomere TERRA molecules in cancer cells, and it can be applied to other cell lines.

Here, we present a protocol to generate cancer cell clones containing a MS2 sequence tag at a single subtelomere. This approach, relying on the MS2-GFP system, enables visualization of the endogenous transcripts of telomeric repeat-containing RNA (TERRA) expressed from a single telomere in living cells.

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in ...

Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have traditionally been attributed to the superficial papillary or lower reticular dermis according to their histological localization. In mouse dermis, papillary and reticular fibroblasts originate from two different lineages with diverging functions regarding physiological and pathological processes and a distinct cell surface marker expression profile by which they can be distinguished.
Importantly, evidence from explant cultures from superficial and lower dermal layers suggest that at least two functionally distinct dermal fibroblasts lineages exist in human skin dermis as well. However, unlike for mouse skin, cell surface markers enabling the discrimination of different fibroblast subsets have not yet been established for human skin. We developed a novel protocol for the isolation of human papillary and reticular fibroblast populations via fluorescence-activated cell sorting (FACS) using the two cell surface markers Fibroblast Activation Protein (FAP) and Thymocyte antigen 1 (Thy1)/CD90. This method enables the isolation of pure fibroblast subsets without in vitro manipulation, which was shown to affect gene expression, thus permitting accurate functional analysis of human dermal fibroblast subsets in regard to tissue homeostasis or disease pathology.

This manuscript describes a FACS-based protocol for isolation of papillary and reticular fibroblasts from human skin. It circumvents in vitro culture which was inevitable with the commonly used isolation protocol via explant cultures. The emanating fibroblast subsets are functionally distinct and display differential gene expression and localization within the dermis.

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Tracking&#160;computer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and wash&#160;buffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.

Use of Single Molecule Fluorescent In Situ Hybridization SM-FISH to Quantify and Localize mRNAs in Murine Oocytes

To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), ...

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often performed at low throughput, it is moreover time-consuming and error-prone, even more so when multiplexing several plasmids. We developed an easy, fast, and accurate method to perform cell transfection in a 384-well plate layout using acoustic droplet ejection (ADE) technology. The nanodispenser device used in this study&#160;is based on this technology and allows precise nanovolume delivery at high speed from a source well plate to a destination one. It can dispense and multiplex DNA and transfection reagent according to a predesigned spreadsheet. Here we present an optimal protocol to perform ADE-based high-throughput plasmid transfection which makes it possible to reach an efficiency of up to 90% and a nearly 100% cotransfection in cotransfection experiments. We extend initial work by proposing a user-friendly spreadsheet-based macro, able to manage up to four plasmids/wells from a library containing up to 1,536 different plasmids, and a tablet-based pipetting guide application. The macro designs the necessary template(s) of the source plate(s) and generates the ready-to-use files for the nanodispenser and tablet-based application. The four-steps transfection protocol involves i) a diluent dispense with a classical liquid handler, ii) plasmid distribution and multiplexing, iii) a transfection reagent dispense by the nanodispenser, and iv) cell plating on the prefilled wells. The described software-based control of ADE plasmid multiplexing and transfection allows even nonspecialists in the field to perform a reliable cell transfection in a fast and safe way. This method enables rapid identification of optimal settings for a given cell type and can be transposed to higher-scale and manual approaches. The protocol eases applications, such as human ORFeome protein (set of open reading frames [ORFs] in a genome) expression or CRISPR-Cas9-based gene function validation, in nonpooled screening strategies.

This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells.

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...