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 arbeidet ble støttet av Deutsche Forschungsgemeinschaft SFB688 (til A.G.).

Musen avl ble godkjent av Regierung Unterfranken, og alle analyser ble utført i strengt samsvar med alle tyske og EUs gjeldende lover og forskrifter om omsorg og bruk av forsøksdyr.
1. coating av være ferdig innen plater med α4β1 Integrin for Ex Utero Allantois Explant Culture
<ol><li>Rekonstituer lyofilisert murine α4β1 integrin på 200 µg/mL i sterilt fosfat-bufret saltvann (PBS). Fortynne til en siste konsentrasjon av 10 µg/mL forvarmes 37 ° c PBS. Pipetter 20 µL/vel a...

<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. 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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 nærvær av serum, som er en rik kilde av Pro lim ekstracellulær matrix molekyler som fibronectin, vil allantois explants lett legge til ulike plast, glass eller filter overflater9,16. Dermed, hvis målet med utført analysen er spesielt undersøke effekten av en genetisk modifisering eller noen andre behandling allantois vedlegg til immobilisert α4β1, er det viktig å blokkere ikke-spesifikk bindende områder (f.eksmed bovin serum albumin) etter be...

Morkaken er uunnværlig for embryonale vekst og utvikling i livmor miljøet. Det utgjør grensesnittet for oksygen, metabolitten og næringsstoffer utveksling mellom fosterets og mors sirkulasjon og også funksjoner som en immunologiske og endokrine organ. Alle endogene eller eksogene krenke som svekker placental utvikling kan føre til intrauterine vekst retardasjon, fosterets tap eller graviditet komplikasjoner, både mus og mennesker1. Mens antall målrettet musen mutasjoner som forårsaker unormale placental fysiologi fortsetter å øke2, med 219 genotyper som er oppført på webområdet musen genomet informatikk (MGI) (http://www.informatics.jax.org/vocab/ mp_ontology / MP:0010038), mange av disse fenotyper er ikke godt forstått fra en mekanistisk synspunkt. I tillegg til genetiske endringer, små molekyl narkotika, monoklonale antistoffer, toksiner, kan patogener, overflødig metabolitter eller ulike miljømessige agenter påvirke morkaken utvikling og funksjon3,4,5 , 6 , 7. ennå, enkel i vitro søk at modellen avgjørende skritt i morkaken utvikling er knappe.
Murine placental utvikling startes i tidlig midgestation, når allantois (utviklingspsykologi forløperen til navlestrengen) kommer fra den bakre enden av embryoet som en knopp som vokser mot plasmocitara8. Chorioadhesive celler i det ytterste laget av allantoic bud megle vedlegget og spredning av allantois på overflaten av plasmocitara (chorioallantoic "fusion"). Plasmocitara kaster deretter inn villi som er blodkar av allantois vokser til labyrinten, det indre placental laget der næringsstoffer og oksygen utveksles mellom tett juxtaposed mors blod sinusoids og embryonale fartøy9 ,10.
Feste allantois til plasmocitara er første og avgjørende skritt i labyrinten formasjon defekter i denne prosessen er blant de vanligste årsakene til embryonale dødelighet i midgestation1. Selv om en rekke musen mutanter har blitt beskrevet der chorioallantoic vedlegg mislykkes oppstår10, og MGI databasen for tiden viser 108 musen mutanter som er preget av unormal chorioallantoic fusion (http:// www.Informatics.JAX.org/vocab/mp_ontology/MP:0002824), intercellulære samspillet mellom vascular celle vedheft molekyl-1 (VCAM-1, på den allantoic mesoderm) og α4β1 integrin (uttrykt på chorion mesothelium, som er avledet fra extraembryonic mesoderm) synes å være uunnværlig for chorioallantoic vedlegg og fusion11,12,13. Immunohistochemical farging av hele-mount vill-type embryo har vist at VCAM-1 er uttrykt i den distale to tredjedeler av allantoic stilken på ca embryonale dag (E) 7.513 (1-4 somite scenen), og at uttrykket er fortsatt høy under chorioallantoic vedlegg og -fusion11,12. Feil på allantoic tilknytning til plasmocitara oppdages vanligvis av histologiske analyser av livmor knopper. Men allantois størrelser er fysiologisk svært variabel i mellom og innenfor søppel av den samme utviklingsstadiet14, og allantois kan bare legge til plasmocitara når den har nådd en tilstrekkelig størrelse å fysisk kontakt. Reflekterer denne naturlige variasjoner, chorioallantoic vedlegg finner sted en gang mellom ~ E 8.0 og E 9.0 i uteroog en statistisk pålitelig evaluering av denne prosessen av histology er derfor avhengig av analyse av et stort antall conceptuses å få nok eksemplarer på den aktuelle utviklingsstadiet.
Her beskriver vi en metode for å vurdere allantoic vedlegg ex utero som er mindre avhengig av allantois størrelse. Vi viser Disseksjon av mouse embryoer og deres allantoides fra uteri gravid mus ~ 8 dager legge coitum (dpc; dpc 0,5 angir deteksjon av en vaginal plugg), og den påfølgende kulturen i allantois explants på immobilisert α4β1 integrins. Denne metoden gjør det mulig for en rask, funksjonelle evaluering av vedlegget og spre virkemåten for flere allantois explants parallelt og påfølgende annen poeng. Protokollen kan brukes til skjermen for effekten av målrettet mutasjoner, narkotika eller ulike miljømessige faktorer på allantois vedlegg 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

Morkaken er viktig for vekst og utvikling av pattedyr embryo. Av denne grunn kan mange genetiske endringer og trolig også miljømessige fornærmelser som forstyrrer morkaken utvikling eller funksjon forårsake tidlig Graviditet i mus og mennesker. Likevel mangler enkel i vitro analyser til skjermen for potensielle virkninger på morkaken formasjon. Her fokusere vi på modellering det første og avgjørende skrittet i morkaken formasjon, som består av feste allantois å plasmocitara. Vi beskriver en metode for å raskt vurdere feste allantoic explants på immobilisert α4β1 Integra som fungerer som en chorio-mimetic substrat. Dette i vitro gir en kvalitativ vurdering av vedlegget og spre virkemåten for flere allantois explants på ulike påfølgende tidspunkt. Protokollen kan brukes å undersøke effekten av målrettet musen mutasjoner, narkotika eller forskjellige miljøfaktorer som har vært knyttet til graviditet komplikasjoner eller fosterets tap på allantois vedlegg ex vivo.

Denne protokollen beskriver en metode for å isolere og explant murine allantoides ex vivoog detaljer kvalitativ vurdering av allantois vedlegg til α4β1 integrin, en kritisk prosess under i vivo chorioallantoic fusion. Representant resultatene som vises i figur 1A--H viser de påfølgende trinnene i allantois isolasjon fra gravid livmor. Mors vev som omgir livmor hornene, som fettvev og fartøy (<strong cl...

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 i vitro analysen modell chorioallantoic vedlegget, det første trinnet i morkaken formasjonen. Protokoll viser murine allantoides på immobilisert α4β1 integrin disseksjon og explant kultur. Allantois vedlegg evalueres mikroskopisk på bestemt tidspunkt.

Disseksjon og Explant kultur Murine Allantois for In Vitro analyse av Allantoic vedlegg

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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