Name: separation of mouse embryonic facial ectoderm and mesenchyme
Uploaded: 2013-04-12T13:00:00
Description: Watch this Scientific Journal Video about Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme at JoVE.com

Forfatterne vil gerne takke Irene Choi til at illustrere embryo hoveder i figur 1 og de øvrige medlemmer af laboratoriet for hjælp og diskussion. Dette arbejde understøttes af NIH tilskud DE012728 (TW)

1. Forbered Dispase II <ol><li> Fremstilles frisk Hepes-pufret saltvand (HBS) (50 mM Hepes / KOH pH 7,4, 150 mM NaCI). Tilsæt 2 ml 0,5 M Hepes / KOH pH 7,4 og 1,2 ml 2,5 M NaCl i 16,8 ml ultrarent H 2 O. </li><li> Lav 10 mg / ml Dispase II. Afvej 0,2 g Dispase II (Roche, Cat # 4942078001) og opløses i 20 ml HBS. Forbered 1,0 ml aliquots i individuelle Eppendorf-rør og opbevares ved -20 ° C for langtidsopbevaring. </li><li> Fortynd 10 mg / ml Dispase II 1:10 i PBS før brug. Sæt den fortyndede Dispase I...

<ol>
	<li>Wilkie, A. O., Morriss-Kay, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+craniofacial+development+and+malformation.">Genetics of craniofacial development and malformation.</a> Nat. Rev. Genet. 2, 458-468 (2001).</li><li>Gritli-Linde, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+2+-+The+Etiopathogenesis+of+cleft+lip+and+cleft+palate:+usefulness+and+caveats+of+mouse+models.">Chapter 2 - The Etiopathogenesis of cleft lip and cleft palate: usefulness and caveats of mouse models.</a> Current Topics in Developmental Biology. 84, 37-138 (2008).</li><li>Schutte, B. C., Murray, 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=The+many+faces+and+factors+of+orofacial+clefts.">The many faces and factors of orofacial clefts.</a> Human Molecular Genetics. 8, 1-7 (1999).</li><li>Chai, Y., Maxson, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+craniofacial+morphogenesis.">Recent advances in craniofacial morphogenesis.</a> Dev. Dyn. 235, 2353-2375 (2006).</li><li>Jiang, R., Bush, J. O., Lidral, A. 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+the+upper+lip:+Morphogenetic+and+molecular+mechanisms.">Development of the upper lip: Morphogenetic and molecular mechanisms.</a> Dev. Dyn. 235, 1152-1166 (2006).</li><li>Reid, B. S., Yang, H., Melvin, V. S., Taketo, M. M., Williams, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectodermal+WNT/&#946;-catenin+signaling+shapes+the+mouse+face.">Ectodermal WNT/&#946;-catenin signaling shapes the mouse face.</a> Developmental Biology. 349, 261-269 (2011).</li><li>Feng, 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=Spatial+and+temporal+analysis+of+gene+expression+during+growth+and+fusion+of+the+mouse+facial+prominences.">Spatial and temporal analysis of gene expression during growth and fusion of the mouse facial prominences.</a> PLoS ONE. 4, e8066 (2009).</li><li>Chia, R., Achilli, F., Festing, M. F., Fisher, E. 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+origins+and+uses+of+mouse+outbred+stocks.">The origins and uses of mouse outbred stocks.</a> Nat. Genet. 37 (11), 1181-1186 (2005).</li><li>Nagai, T., Aruga, J., Takada, S., 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+expression+of+the+mouse+Zic1,+Zic2,+and+Zic3+gene+suggests+an+essential+role+for+Zic+genes+in+body+pattern+formation.">The expression of the mouse Zic1, Zic2, and Zic3 gene suggests an essential role for Zic genes in body pattern formation.</a> Developmental Biology. 182, 299-313 (1997).</li><li>Britsch, S., 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+transcription+factor+Sox10+is+a+key+regulator+of+peripheral+glial+development.">The transcription factor Sox10 is a key regulator of peripheral glial development.</a> Genes Dev. 15 (1), 66-78 (2001).</li></ol>

Denne protokol giver en enkel metode til at skille embryonale muse facial ektoderm og mesenkym baseret på en initial Dispase II behandlingstrin. I tidligere undersøgelser er der udført dissektion af ansigtets Fremspring før Dispase II behandling, men vi konsekvent har vist sig, at mesenkym bliver &quot;klæbrige&quot; og vanskeligere at manipulere. Vores nye protokol undgår dette problem. Kombinationen Dispase II behandling under forsigtig fysisk kraft ved at pipettere op og ned gør adskillelse af ektoderm og mese...

<table>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <td>Dispase II (neutral protease, grade II)</td>
 <td>Roche</td>
 <td>4942078001</td>
 <td>Make 10 mg/ml Dispase II stock solution in HBS. Aliquot and store at -20 &deg;C . </td>
 </tr>
 <td>RNAlater</td>
 <td>Ambion</td>
 <td>AM7021</td>
 <td>&nbsp;</td>
 </tr>
</table>

separation of mouse embryonic facial ectoderm and mesenchyme

Orofacial kløfter er de mest hyppige kraniofaciale defekter, som påvirker 1,5 ud af 1.000 nyfødte på verdensplan 1,2. Orofaciale clefting er forårsaget af unormal facial udvikling 3. I human og mus, initial vækst og mønsterdannelse af ansigtet er afhængig af flere små knopper af væv, ansigtets Fremspring 4,5. Ansigtet er afledt af seks store Fremspring: parrede frontal nasale processer (FNP), kaebe Fremspring (MXP) og mandiblens fremspring (MDP). Disse fremspring består af hævelse af mesenkym der er indkapslet i et overliggende epitel. Undersøgelser hos flere arter har vist, at signalering krydstale mellem facial ektoderm og mesenkym er kritisk for at forme flade 6. Alligevel er mekanistiske detaljer vedrørende generne involveret i disse signal-relæer mangler. En måde at få en bred forståelse af genekspression, transkriptionsfaktor bindende, og kromatin mærker i forbindelse med udvikping facial ektoderm og mesenkym er at isolere og karakterisere de adskilte væv rum. Her præsenteres en fremgangsmåde til adskillelse facial ektoderm og mesenkym på fosterdag (E) 10,5, en kritisk udviklingsstadiet i mus facial formation, der går forud for smeltning af Fremspring. Vores metode er tilpasset fra den tilgang, vi tidligere har brugt til dissekering ansigtet Fremspring 7. I denne tidligere undersøgelse havde vi ansat indavlede C57BL / 6 mus som denne stamme er blevet en standard for genetik, genomforskning og i ansigtet morfologi 8. Her dog, på grund af de mere begrænsede mængder af væv til rådighed, har vi udnyttet den udavlet CD-1-stamme, der er billigere at købe, mere robust for dyrehold, og har tendens til at producere flere embryoner (12-18) per kuld end nogen indavlet musestamme 8. Efter embryo isolation blev neutral protease Dispase II til behandling af hele embryo. Derefter blev facial Fremspring dissekereed ud, og den facial ektoderm blev separeret fra mesenkym. Denne metode holder både ansigts ektoderm og mesenkym intakt. Prøverne opnået under anvendelse af denne metode kan anvendes til teknikker, herunder proteindetektion, chromatin immunoprecipitation (chip) assay, microarray undersøgelser, og RNA-seq.

Efter Dispase II behandling tendens til, at ectoderm af embryonet til at være løst. Ansigtets fremspring skal være intakt efter dissektion (figur 2A). Det isolerede facial ektoderm er klar og fri for mesenkym væv (figur 2B). For at bestemme effektiviteten af protokollen og for at opdage krydskontaminering vi analyseret for forhjerne udtrykte Zic3 9, ectodermal specifik CDH1 5 og mesenchymal specifik sox10 10 under anvendelse af revers transkriptase P...

Watch this Scientific Journal Video about Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme at JoVE.com

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

En protokol til adskillelse af foster facial ektoderm og mesenkym er beskrevet. Vi bruger Dispase II til behandling af hele embryoner først, dissekere hele ansigtet Fremspring ud, og derefter adskille facial ektoderm og mesenkym.

Adskillelse af muse embryonale Facial ektoderm og mesenkym

 Orofacial clefts are the most frequent  craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal ...

Research

JoVE Journal

Biology

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic ...

Electroporation of Craniofacial Mesenchyme

Elektroporation af kraniofaciale Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

Udarbejdelse af muse embryonale fibroblastceller anvendes til dyrkning af menneskelige embryoner og induceret pluripotente stamceller

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

A System for ex vivo Culturing of Embryonic Pancreas

A System for ex vivo Culturing of Embryonic Pancreas

Et system til<em&gt; Ex vivo</em&gt; Dyrkning af embryonale Pancreas

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

En Eksplantation Assay for vurdering Cellulær Behavior af Cranial mesenkym

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...