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

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

Allmän information om grodor och salamandrar, liksom källor till djur, kan hittas på följande webbplatser: Xenbase (http://www.xenbase.org) och Sal-Site (http://www.ambystoma.org). Detta protokoll följer riktlinjerna för djurskötsel av avdelningen av embryologi av Carnegie Institution for Science. 1. Lösningar <ol><li> Göra 10 L &quot;groda vatten&quot;: Tillsätt 10 ml 1 M CaCl2 och 10 ml 1 M NaHCOs 3 till 10 L avjoniserat eller avklorerat H2O und...

<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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lariat+intronic+RNAs+in+the+cytoplasm+of+Xenopus+tropicalis+oocytes.">Lariat intronic RNAs in the cytoplasm of Xenopus tropicalis oocytes.</a> RNA. 20, 1476-1487 (2014).</li><li>Duryee, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+nuclei+and+non-mitotic+chromosome+pairs+from+frog+eggs.">Isolation of nuclei and non-mitotic chromosome pairs from frog eggs.</a> Arch. Exp. Zellforsch. 19, 171-176 (1937).</li></ol>

De första observationerna av &quot;levande&quot; LBC från hand isolerade GVS av grodor och salamandrar gjordes nästan 80 år sedan av den amerikanska biologen William Duryee, 19 före införandet av faskontrast och DIC mikroskopi, innan fluorescerande immunfärgning, och innan FISH. Fördelarna med LBC för att undersöka uppgifter om kromosomstrukturen och transkription på individuell gennivå krävs utveckling av tekniker för att fästa LBC till objektglas, för att fixera dem på ett verklighetstroget...

De flesta ryggradsdjur, med det anmärkningsvärda undantaget av pungdjur och moderkakan däggdjur, producerar stora yolky ägg. Trots deras ibland enorma storlek, dessa ägg är enstaka celler som når sin slutliga dimension samtidigt i äggstocken av den kvinnliga. Äggstocks ägg kallas ägg och var och en innehåller vanligtvis en enda gigantisk kärna, känd sedan början av 19-talet som germinala vesikeln eller helt enkelt GV. 1 Oocyter av den gemensamma laboratorie grodor, Xenopus laevis och Xenopus tropic, nå en maximal diameter på 1,2 mm och 0,8 mm respektive (Figur 1). De GVS från mogna oocyter från dessa två grodor är 0,3 - 0,4 mm i diameter (fig 2, 3). Salamandrar har typiskt ännu större oocyter och GVS. Fullt mogna ägg i den mexikanska axolotl, Ambystoma mexicanum, är mer än 2 mm i diameter och GV är ca 0,5 mm. Således är dessa kärnor är lätt synliga för blotta ögat och kanmanipuleras på många sätt som är omöjligt med kärnorna hos typiska somatiska celler. Lika anmärkningsvärt är den gigantiska storlek kromosomerna inom GV, ett faktum redan erkänt i slutet av 19-talet. Individuella kromosomerna hos Ambystoma och andra salamandrar kan vara upp till 1 mm i längd (Figurerna 4, 5). De av Xenopus är betydligt mindre, men med längder upp till 100 pm eller mer, de dvärg typiska somatiska kromosomer de flesta organismer. Ett viktigt inslag i äggcell kromosomer är deras extraordinära transkriptionsaktivitet, vilket leder till en av sina mest karakteristiska morfologiska egenskaper - hundratals parade sido slingor (Figur 5). Varje slinga består av en eller ett fåtal transkriptionsenheter som aktivt syntetiserar RNA. Slingorna ger äggcellen kromosomer en suddig utseende, vilket ledde till namnet &quot;lampbrush&quot; kromosom efter deras ytligalikhet med de borstar som används i äldre tider för att rengöra fotogenlampa skorstenar. 2 Fokus i denna uppsats är om användningen av isolerade GVS att studera LBC och kärnorgan (nukleolerna, histon locus organ och prickar). Två ganska olika tekniker kommer att beskrivas. I den första, mer vanlig teknik, är GVS isoleras i en koksaltlösning med användning av jeveleraraffärer pincett, i korthet sköljdes för att avlägsna vidhäftande äggula, och kärnhöljet avlägsnas, återigen med jeveleraraffärer pincett. De gelatin innehåll, innehåller LBC och kärnorgan, får sedimentera på en glasobjektglas eller täck. Sådana preparat kan undersökas direkt genom faskontrast eller DIC mikroskopi. Alternativt kan beredningar centrifugeras för att fästa LBC och organeller till bilden eller täck. Sådana preparat kan sedan bearbetas för detaljerad molekylär analys av nukleinsyror och proteiner, främst genom immunofluorescens och fluorescent in situ-hybridisering (FISH). 3-7 En andra teknik innefattar isolering av GV i mineralolja. 8 Olje isolerade GVS förblir transkriptionellt aktiv i många timmar och är potentiellt användbara för studier där man vill kärn innehållet att vara så naturtrogen som möjligt. 9,10 Eftersom brytningsindex för den nukleära &quot;sav&quot; är nära den för de-LBC och andra kärnorganeller (Figur 3), kan mikroskopiska tekniker vara en utmaning med olje isolerade GVS. Slutligen, på grund av sin storlek och enkel hantering, GSV är idealiskt material för studier på kärnhöljet. Den kärnpor komplex beskrevs först från elektronmikroskopstudier på amfibie GV kuvert 11 och nyare Superresolution observationer har använt samma 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

Vi beskriver metoder för att studera den gigantiska transkription aktiva lampbrush kromosomer (LBC) finns i äggcellen, eller unlaid ägg, grodor och salamandrar. Enskilda LBC kan vara upp till 1 mm i längd och de bor i en gigantisk kärnan själv upp till 0,5 mm i diameter. Den stora storleken på kromosomerna medger motstycke observationer av aktiva gener genom ljus optisk mikroskopi, men på samma gång speciella tekniker erfordras för att isolera kärnan, avlägsna kärnhöljet, och sprida kromosomerna på ett objektglas. Äggcellen nucleus, även kallad den germinala vesikeln (GV), isoleras i ett medium som tillåter partiell gelning av den nukleära aktin och bevarar den ömtåliga strukturen av LBC. Detta steg utförs manuellt under ett dissektionsmikroskop med användning av guldsmeder pincett. Därefter kärnhöljet avlägsnas, återigen manuellt med jeveleraraffärer pincett. De nukleära innehållet snabbt överföras till ett medium som disperses aktin gelen och tillåter de oskadade LBC sedimentera på ett objektglas. Vid denna punkt LBC och andra kärnorgan kan ses av faskontrast eller differentiell interferenskontrast mikroskopi, även om finare detaljer skyms av Brownsk rörelse. För hög upplösning mikroskopisk observation eller molekylär analys är hela beredningen centrifuger att fästa den känsliga LBC fast bilden. En kort fixering i paraformaldehyd följs sedan av immunofluorescerande färgning eller in situ hybridisering. LBC är i ett transkription aktivt tillstånd och deras enorma storlek medger molekylär analys på individ gennivå med hjälp av konfokal eller super-upplösning mikroskopi.

För att undersöka jätte lampbrush kromosomer man börjar genom att isolera ägg från en groda eller salamander. Figur 1 visar en grupp av mogna oocyter i en buffrad salinlösning efter avlägsnande från äggstocken hos groda, Xenopus. Sådana ägg kvar i gott skick för dagar vid rumstemperatur. Kärnan (eller germinal vesikel) avlägsnas sedan från en oocyt med jeveleraraffärer pincett, antingen i en koksaltlösning (figur 2) eller i olj...

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

Vi presenterar enkla tekniker för att isolera jättetranskription aktiva lampbrush kromosomer från levande oocyter av grodor och salamandrar. Vi beskriver hur man följa dessa kromosomer &quot;levande&quot; av faskontrast eller differentiell interferenskontrast, och hur man rättar dem för fluorescent in situ hybridisering eller immunofluorescerande färgning.

Isolering av Giant lampbrush kromosomer från Living oocyter av grodor och salamandrar

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

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

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

Provberedning och analys av RNASeq-baserade gen uttryck Data från zebrafisk

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

Formaldehyd-assisted isolering av reglerande element till åtgärd kromatin tillgänglighet i däggdjursceller

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

Använda mus oocyter att bedöma mänskliga geners funktion under meios jag

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

Isolering, karakterisering och mikroRNA-baserad genetisk modifiering av mänskliga dentala follikeln stamceller

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

Generation av Cancer Cell kloner att visualisera Telomeric Repeat-innehållande RNA TERRA uttryckt från en enda telomeren i levande celler

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

Isolering av papillär och retikulära fibroblaster från Human Skin genom Fluorescence-aktive rad cell sortering

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

Sekvensering av mRNA från helblod med Nanopore sekvensering

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

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

Användning av enda molekyl fluorescerande i Situ hybridisering (SM-fisk) för att kvantifiera och lokalisera mRNA i murina oocyter

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

Hög genomströmning DNA plasmid Multiplexing och transfektion med akustisk Nanodispenseringsteknik

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