Name: dissection and explant culture of murine allantois for the in vitro analysis of allantoic attachment
Uploaded: 2018-01-13T13:00:00
Description: Watch this Scientific Journal Video about Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment at JoVE.com

Dette arbejde blev støttet af Deutsche Forschungsgemeinschaft SFB688 (til A.G.).

Musen avl blev godkendt af Regierung Unterfranken, og alle analyser blev udført i nøje overensstemmelse med alle tyske og Europæiske Union gældende love og bestemmelser vedrørende pleje og anvendelse af forsøgsdyr.
1. belægning af mikrotiter plader med α4β1 Integrin for Ex Utero Allantois eksplantat kultur
<ol><li>Rekonstruere den frysetørrede murine α4β1 integrin på 200 µg/mL i sterilt fosfatbufferet saltopløsning (PBS). Fortyndes til en slutkoncentration på 10 µg/...

<ol>
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E., Downs, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+murine+allantois:+emerging+paradigms+in+development+of+the+mammalian+umbilical+cord+and+its+relation+to+the+fetus.">The murine allantois: emerging paradigms in development of the mammalian umbilical cord and its relation to the fetus.</a> Genesis. 45 (5), 237-258 (2007).</li><li>Arora, R., Papaioannou, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+murine+allantois:+a+model+system+for+the+study+of+blood+vessel+formation.">The murine allantois: a model system for the study of blood vessel formation.</a> Blood. 120 (13), 2562-2572 (2012).</li><li>Watson, E. D., Cross, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+structures+and+transport+functions+in+the+mouse+placenta.">Development of structures and transport functions in the mouse placenta.</a> Physiology (Bethesda). 20, 180-193 (2005).</li><li>Yang, J. T., Rayburn, H., Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+events+mediated+by+alpha+4+integrins+are+essential+in+placental+and+cardiac+development.">Cell adhesion events mediated by alpha 4 integrins are essential in placental and cardiac development.</a> Development. 121 (2), 549-560 (1995).</li><li>Gurtner, G. C., 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=Targeted+disruption+of+the+murine+VCAM1+gene:+essential+role+of+VCAM-1+in+chorioallantoic+fusion+and+placentation.">Targeted disruption of the murine VCAM1 gene: essential role of VCAM-1 in chorioallantoic fusion and placentation.</a> Genes &amp; development. 9 (1), 1-14 (1995).</li><li>Kwee, L., 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=Defective+development+of+the+embryonic+and+extraembryonic+circulatory+systems+in+vascular+cell+adhesion+molecule+(VCAM-1)+deficient+mice.">Defective development of the embryonic and extraembryonic circulatory systems in vascular cell adhesion molecule (VCAM-1) deficient mice.</a> Development. 121 (2), 489-503 (1995).</li><li>Downs, K. M., Gardner, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+investigation+into+early+placental+ontogeny:+allantoic+attachment+to+the+chorion+is+selective+and+developmentally+regulated.">An investigation into early placental ontogeny: allantoic attachment to the chorion is selective and developmentally regulated.</a> Development. 121 (2), 407-416 (1995).</li><li>Heyne, G. W., 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=A+simple+and+reliable+method+for+early+pregnancy+detection+in+inbred+mice.">A simple and reliable method for early pregnancy detection in inbred mice.</a> J Am Assoc Lab Anim Sci. 54 (4), 368-371 (2015).</li><li>Downs, K. M., Temkin, R., Gifford, S., McHugh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+the+murine+allantois+by+allantoic+explants.">Study of the murine allantois by allantoic explants.</a> Dev Biol. 233 (2), 347-364 (2001).</li><li>Hou, W., Sarikaya, D. P., Jerome-Majewska, 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=Ex+vivo+culture+of+pre-placental+tissues+reveals+that+the+allantois+is+required+for+maintained+expression+of+Gcm1+and+Tpbpalpha.">Ex vivo culture of pre-placental tissues reveals that the allantois is required for maintained expression of Gcm1 and Tpbpalpha.</a> Placenta. 47, 12-23 (2016).</li><li>Zeigler, B. M., 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=The+allantois+and+chorion,+when+isolated+before+circulation+or+chorio-allantoic+fusion,+have+hematopoietic+potential.">The allantois and chorion, when isolated before circulation or chorio-allantoic fusion, have hematopoietic potential.</a> Development. 133 (21), 4183-4192 (2006).</li><li>Zaidel-Bar, R., Itzkovitz, S., Ma'ayan, A., Iyengar, R., Geiger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+atlas+of+the+integrin+adhesome.">Functional atlas of the integrin adhesome.</a> Nature cell biology. 9 (8), 858-867 (2007).</li></ol>

I nærværelse af serum, der er en rig kilde af Pro selvklæbende ekstracellulære matrix molekyler såsom fibronektin, vil allantois explants let tillægger forskellige plast, glas eller filter overflader9,16. Derfor, hvis formålet med udføres analysen er specifikt undersøge effekten af en genetisk modifikation eller enhver anden behandling på allantois tilknytning til ubevaegeligt α4β1, er det kritisk at blokere ikke-specifik binding websteder (f.eks....

Moderkagen er uundværlige for embryonale vækst og udvikling i den uterine miljø. Det udgør grænsefladen for ilt, metabolit, og næringsstof udveksling mellem føtal og maternel omsætning, og også funktioner som en endokrine og immunologiske orgel. En endogen eller eksogen fornærmelse, der forringer placenta udvikling kan føre til intrauterin væksthæmning, føtal tab eller graviditetskomplikationer, både mus og mennesker1. Mens antallet af målrettede mus mutationer, der forårsager unormal placenta fysiologi fortsætter med at stige2, med 219 genotyper i øjeblikket opført på webstedet mus genom Informatik (MGI) (http://www.informatics.jax.org/vocab/ mp_ontology / MP:0010038), mange af disse fænotyper er ikke velbeskrevet fra et mekanisk synspunkt. Ud over genetiske ændringer, lille molekyle narkotika, monoklonale antistoffer, toksiner, kan patogener, overskydende metabolitter eller forskellige miljømæssige agenter påvirke moderkagen udvikling og funktion3,4,5 , 6 , 7. endnu, enkel in vitro- undersøgelser, at model kritiske trin i moderkagen udvikling er knappe.
Murine placenta udvikling initieres i tidlige midgestation, når allantois (den udviklingsmæssige forløber for navlestrengen) opstår fra den bageste ende af fosteret som et bud, der vokser mod chorion8. Chorioadhesive celler i de yderste lag af allantois bud mægle den vedhæftede fil og spredning af allantois på overfladen af chorion (chorioallantoic "fusion"). Chorion folder efterfølgende i villi, hvori Vaskulaturen af allantois vokser til at danne labyrinten, den inderste placenta lag hvor næringsstoffer og ilt udveksles mellem tæt sidestillede maternelle blod sinusoids og embryonale fartøjer9 ,10.
Allantois tilknytning til chorion er det første og afgørende skridt i labyrint dannelse, og mangler i denne proces er blandt de mest almindelige årsager til embryonale dødelighed i midgestation1. Selv om en række mus mutanter har været beskrevet hvor chorioallantoic vedhæftet undlader at forekomme10, og MGI databasen i øjeblikket viser 108 mus mutanter, der er kendetegnet ved unormal chorioallantoic fusion (http:// www.Informatics.Jax.org/vocab/mp_ontology/MP:0002824), intercellulære samspillet mellem vaskulære celle vedhæftning molekyle-1 (VCAM-1, udtrykt på den allantois mesoderm) og α4β1 integrin (udtrykt på chorion mesothelium, som er afledt af extraembryonic mesoderm) synes at være uundværlig for chorioallantoic vedhæftet fil og fusion11,12,13. Immunhistokemisk farvning af hele-mount vildtype embryoner har vist at VCAM-1 er udtrykt i den distale totredjedele af allantois stokken på ca embryonale dag (E) 7,513 (1-4 somite fase), og at dens udtryk er stadig høj under chorioallantoic vedhæftet fil og -fusion11,12. Manglende allantois tilknytning til chorion er typisk opdaget af histologiske analyser af livmoderen knopper. Men allantois størrelser er fysiologisk meget varierende i mellem og selv inden for kuld af den samme udviklingsstadiet14, og allantois kan kun knytte til chorion når det har nået en tilstrækkelig størrelse til at gøre fysisk kontakt. Afspejler denne naturlige variabilitet, chorioallantoic udlæg finder sted engang imellem ~ E 8.0 og E 9.0 i uteroog en statistisk pålidelig evaluering af denne proces af histologi er derfor afhængige af analysen af et stort antal conceptuses at få nok prøver på den passende udviklingsstadiet.
Her, beskriver vi en metode til at vurdere allantois vedhæftede ex utero som er mindre afhængige af allantois størrelse. Vi demonstrere dissektion af musen embryoner og deres allantoides fra livmoder af gravide mus ~ 8 dage post coitum (dpc; dpc 0,5 udpeger påvisning af en vaginal plug), og den efterfølgende kultur af allantois explants på immobiliserede α4β1 integriner. Denne metode giver mulighed for en hurtig, funktionel evaluering af den vedhæftede fil og sprede adfærd af flere allantois explants parallelt og på forskellige successive tidspunkter. Protokollen kan anvendes til at screene for målrettede mutationer, narkotika eller forskellige miljøfaktorers indvirkning på allantois vedhæftet fil ex vivo.

<table border="0" cellpadding="0" cellspacing="0" width="1607">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:319px;">C57BL/6J wildtype mice</td>
			<td style="width:256px;">The Jackson Laboratory</td>
			<td style="width:119px;">Stock No:&#160;000664</td>
			<td style="width:915px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70 % (v/v) Ethanol</td>
			<td style="width:256px;">VWR International</td>
			<td>930031006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard pattern forceps</td>
			<td style="width:256px;">Fine Science Tools</td>
			<td>11000-12</td>
			<td style="width:915px;">Forceps used to open the abdominal cavity.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Micro dissecting forceps</td>
			<td style="width:256px;">Fine Science Tools</td>
			<td style="width:119px;">11254-20</td>
			<td>Dumont # 5 medical biology forcepts with fine, sharp tip.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Fine scissors&#160;</td>
			<td style="width:256px;">Fine Science Tools</td>
			<td>14094-11</td>
			<td>Straight blades.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spring scissors&#160;</td>
			<td style="width:256px;">Fine Science Tools</td>
			<td>15012-12</td>
			<td>Noyes spring scissors with 14 mm blades for the dissection of uterus buds.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Microspoon</td>
			<td style="width:256px;">Carl Roth</td>
			<td>AT18.1</td>
			<td>5 mm Spoon diameter.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Microtiter plates</td>
			<td style="width:256px;">ibidi</td>
			<td style="width:119px;">81501</td>
			<td>We use uncoated, sterile angiogenesis slides (internal volume 50 &#181;L) with a hydrophobic surface that is not tissue-culture treated to reduce non-a4b1-mediated binding to plastic.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">10 cm dish</td>
			<td style="width:256px;">Nunc</td>
			<td style="width:119px;">150350</td>
			<td>Sterile tissue culture dishes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">3.5 cm dish</td>
			<td style="width:256px;">Nunc</td>
			<td style="width:119px;">150288</td>
			<td>Sterile tissue culture dishes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Large orifice pipette tips&#160;</td>
			<td style="width:256px;">Biozym</td>
			<td style="width:119px;">VT0140X</td>
			<td>Low binding pipette tips, 200 &#181;L.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">a4b1 integrin</td>
			<td style="width:256px;">R&#38;D Systems</td>
			<td style="width:119px;">6054-A4</td>
			<td>Murine a4b1 integrin recombinantly expressed and purified from a Chinese hamster ovary cell line.</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:319px;">Dulbecco&#39;s Phosphate Buffered Saline (PBS)</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td>Without Ca2+ and Mg2+.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:319px;">Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td style="width:256px;">PAN Biotech</td>
			<td style="width:119px;">P04-03600</td>
			<td style="width:915px;">The formulation contains 4.5 g/L glucose, 110 mg/L sodium pyruvate and 3.7 g/L NaHCO3 but no L-glutamine, which is added separately.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:319px;">Penicillin/Streptomycin</td>
			<td style="width:256px;">Gibco (ThermoFisher Scientific)</td>
			<td style="width:119px;">15140-122</td>
			<td>100 x (10,000 U/mL Penicillin and 10,000 &#181;g/mL streptomycin) stock solution.&#160;Prepare and store aliquots at -20 &#176;C to avoid freeze/thaw.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">L-Glutamine</td>
			<td style="width:256px;">PAN Biotech</td>
			<td style="width:119px;">P04-80100</td>
			<td>100 x (200 mM) Stock solution. Prepare and store aliquots at -20 &#176;C to avoid freeze/thaw.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Fetal bovine serum&#160;</td>
			<td style="width:256px;">Biochrom</td>
			<td style="width:119px;">S 0115</td>
			<td>We use heat-inactivated FBS (heated to 56 &#176;C for 30 min with mixing to inactivate complement).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Bovine serum albumin (BSA)</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td style="width:119px;">A7030</td>
			<td>We use protease- and fatty acid-free albumin. Prepare a 0.1 % (w/v) solution in DMEM. Sterile filter the solution through a disposable cell culture filter with a 0.22 &#181;m pore size and low protein-binding membrane, and store aliquots at -20 &#176;C.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">para-Formaldehyde</td>
			<td style="width:256px;">Carl Roth</td>
			<td>0335.2</td>
			<td style="width:915px;">To make a formaldehyde solution in PBS, weigh para-formaldehyde powder in a ventilated hood, and add it to PBS preheated to ca. 60 &#176;C in a beaker on a stir plate. Add 1 N NaOH dropwise until solution clears. Let solution cool to room temperature, and filter through 0.22 mm filter syringe. Adjust pH with diluted HCl to ca. 6.9, and adjust to final volume with PBS. We aliquot and freeze the solution, but it can also be stored at 4 &#176;C for approximately four weeks.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Triton X-100</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td style="width:119px;">X100</td>
			<td>Use wide-orifice pipette tips to handle undiluted Triton X-100.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Normal goat serum</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td>G9023</td>
			<td>To block non-specific binding of antibodies in immunofluorescence analyses.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">a-CD31 Antibodies</td>
			<td style="width:256px;">BD Biosciences</td>
			<td>550274</td>
			<td>Purified rat anti-mouse monoclonal antibodies, clone MEC 13.3.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Secondary goat anti-rat antibodies</td>
			<td style="width:256px;">Thermo Fisher</td>
			<td>A-11006</td>
			<td>Goat anti-rat IgG (heavy and light chains), cross-adsorbed, fluorescently labeled with Alexa Fluor 488. Other fluorescent labels are also possible.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Mowiol 4-88</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td>81381</td>
			<td>Mounting medium for cytochemistry. MW ~31,000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Labovert&#160;</td>
			<td style="width:256px;">Leitz</td>
			<td style="width:119px;">Labovert&#160;</td>
			<td>Inverted phase contrast microscope equipped with a 4 x and 10 x objective for somite pair counting. Other phase contrast microscopes (such as those that are routinely used in tissue culture) are also suitable.&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">M80</td>
			<td style="width:256px;">Leica Microsystems</td>
			<td style="width:119px;">M80</td>
			<td>Stereomicroscope with 8 : 1 zoom range (yielding a magnification between 7.5 x and 60 x) for embryo and allantois dissection.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">DM4000B</td>
			<td style="width:256px;">Leica Microsystems</td>
			<td style="width:119px;">DM4000B</td>
			<td>Microscope system for histology with LED illumination. We use HCX PL FLUOTAR 5 x/0.15 and HC PL FLUOTAR 10 x/0.30 objectives. Other microscopes equipped phase contrast and fluorescence optics can be used to score allantois attachment.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Digital camera</td>
			<td style="width:256px;">JVC</td>
			<td style="width:119px;">KY-F75U</td>
			<td>3-CCD digital capture camera attached to the DM4000B LED microscope. We use Diskus software (Hilgers) to operate both the microscope and the camera.</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:319px;">Confocal microscope</td>
			<td style="width:256px;">Leica Microsystems</td>
			<td style="width:119px;">SP5</td>
			<td style="width:915px;">Confocal microscope for higher-resolution imaging of endothelia in the vascular plexus of allantois explants. Immunofluorescence images were taken with a 40 x objective.</td>
		</tr>
	</tbody>
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dissection and explant culture of murine allantois for the in vitro analysis of allantoic attachment

Moderkagen er afgørende for vækst og udvikling af pattedyr embryoner. Af denne grund, kan mange genetiske ændringer og sandsynligvis også miljømæssige fornærmelser, som forstyrrer moderkagen udvikling eller funktion forårsage tidlig graviditet tab i mus og mennesker. Ikke desto mindre, simple in vitro- assays til at screene for potentielle virkninger på moderkagen dannelse mangler. Her fokuserer vi på modellering det første og afgørende skridt i moderkagen dannelse, som består af allantois tilknytning til chorion. Vi beskriver en metode til hurtigt at vurdere udlæg i allantois explants på immobiliserede α4β1 Integra, som fungerer som en chorio-mimetiske substrat. Denne in vitro- metode muliggør en kvalitativ vurdering af den vedhæftede fil og sprede adfærd af flere allantois explants på forskellige på hinanden følgende tidspunkter. Protokollen kan bruges til at undersøge effekten af målrettede mus mutationer, narkotika eller forskellige miljømæssige faktorer, der har været knyttet til graviditetskomplikationer eller føtal tab på allantois vedhæftet fil ex vivo.

Denne protokol beskriver en metode til at isolere og explant murine allantoides ex vivo, og oplysninger om den kvalitative vurdering af allantois vedhæftet fil til α4β1 Integra, en kritisk proces under i vivo chorioallantoic fusion. Repræsentative resultaterne vist i figur 1A-H demonstrere de successive trin allantois isolation fra den gravide uterus. Maternelle væv, der omgiver den uterine horn, fedtv?...

Watch this Scientific Journal Video about Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment at JoVE.com

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

Vi beskriver en i vitro assay til model chorioallantoic vedhæftet fil, det første skridt i moderkagen dannelse. Protokollen viser murine allantoides på immobiliserede α4β1 integrin dissektion og eksplantat kultur. Allantois udlæg er evalueret mikroskopisk på forud fastsatte tidspunkter.

Dissektion og eksplantat kultur af Murine Allantois til In Vitro -analyse af allantois vedhæftet fil

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental ...

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Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

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