Name: loss- and gain-of-function approach to investigate early cell fate determinants in preimplantation mouse embryos
Uploaded: 2016-06-06T13:00:00
Description: Watch this Scientific Journal Video about Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos at JoVE.com

Forfatterne vil også gerne anerkende dr Xiong gruppe på Georgia Health Sciences University for produktion og deling af konstruktionerne for neogenin cDNA og shRNA vektorer. Denne undersøgelse blev støttet af tilskud fra Basic Science Research Program (2013R1A1A4A01012572) finansieret af den koreanske Grundforskningsfond og ved en forskningsbevilling fra Sahmyook University.

BEMÆRK: Alle procedurer for dyreavl og bekymringer blev udført i overensstemmelse med IACUC forskrifter Sahmyook University, Seoul, Sydkorea. 1. superovulation, Avl og æggelederen Isolation <ol><li> Oprethold indavlede C57BL / 6-mus under følgende betingelser: 22 ± 3 ° C, ~ 60% fugtighed, 12 timers lys / mørke-cyklus, og vand og foder ad libitum. </li><li> Inducere superovulation af 3-5 uger gamle hunmus ved en intraperitoneal injektion af 5 IU drægtig hoppe-serum gonadotropin (PMSG) ved 0,1 ml / dyr e...

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	<li>Yamanaka, Y., Ralston, A., Stephenson, R. O., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+and+molecular+regulation+of+the+mouse+blastocyst.">Cell and molecular regulation of the mouse blastocyst.</a> Dev. Dyn. 235 (9), 2301-2314 (2006).</li><li>Sasaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+trophectoderm+fate+specification+in+preimplantation+mouse+development.">Mechanisms of trophectoderm fate specification in preimplantation mouse development.</a> Dev. Growth Differ. 52 (3), 263-273 (2010).</li><li>Nishioka, N., Yamamoto, S., Kiyonari, H., Sato, H., Sawada, A., Ota, 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=Tead4+is+required+for+specification+of+trophectoderm+in+pre-implantation+mouse.">Tead4 is required for specification of trophectoderm in pre-implantation mouse.</a> Mech. Dev. 125 (3-4), 270-283 (2008).</li><li>Ovitt, C. E., Sch&#246;ler, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+biology+of+Oct-4+in+the+early+mouse+embryo.">The molecular biology of Oct-4 in the early mouse embryo.</a> Mol. Hum. Reprod. 4 (11), 1021-1031 (1998).</li><li>Stein, P., Schindler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Oocyte+Microinjection,+Maturation+and+Ploidy+Assessment.">Mouse Oocyte Microinjection, Maturation and Ploidy Assessment.</a> J. Vis. Exp. (53), e2851 (2011).</li><li>Shin, M. R., Cui, X. S., Jun, J. H., Jeong, Y. J., Kim, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+mouse+blastocyst+genes+that+are+down+regulated+by+double-stranded+RNA-mediated+knockdown+of+Oct-4+expression.">Identification of mouse blastocyst genes that are down regulated by double-stranded RNA-mediated knockdown of Oct-4 expression.</a> Mol. Reprod. Dev. 70 (4), 390-396 (2005).</li><li>Khang, I., Sonn, S., Park, J. -. H., Rhee, K., Park, D., Kim, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+epithin+in+mouse+preimplantation+development:+Its+role+in+compaction.">Expression of epithin in mouse preimplantation development: Its role in compaction.</a> Dev. Biol. 281, 134-144 (2005).</li><li>Prieto, D., Aparicio, G., Machado, M., Zolessi, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+the+DNA-Specific+Stain+Methyl+Green+in+the+Fluorescent+Labeling+of+Embryos.">Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos.</a> J. Vis. Exp. (99), e52769 (2015).</li><li>Lee, J. H., Choi, S. S., Kim, H. W., Xiong, W. C., Min, C. K., Lee, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neogenin+as+a+receptor+for+early+cell+fate+determination+in+preimplantation+mouse+embryos.">Neogenin as a receptor for early cell fate determination in preimplantation mouse embryos.</a> PLoS One. 9 (7), e101989 (2014).</li><li>Gordon, J. W., Scangos, G. A., Plotkin, D. J., Barbosa, J. A., Ruddle, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+transformation+of+mouse+embryos+by+microinjection+of+purified+DNA.">Genetic transformation of mouse embryos by microinjection of purified DNA.</a> Proc. Natl. Acad. Sci. USA. 77 (12), 7380-7384 (1980).</li><li>Brinster, R. L., Chen, H. Y., Trumbauer, M. E., Yagle, M. K., Palmiter, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+the+efficiency+of+introducing+foreign+DNA+into+mice+by+microinjecting+eggs.">Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs.</a> Proc. Natl. Acad. Sci. USA. 82, 4438-4442 (1985).</li></ol>

I nærværende protokol, vi demonstrere nye metoder til mikroinjektion af genetisk materiale, enten cDNA eller shRNA, ind i de to-PN museembryoer at udforske rolle neogenin i begyndelsen celledifferentiering under mus embryogenese. Genetisk modifikation af præimplantations embryoer er en kraftfuld teknik at afdække centrale oplysninger om underliggende molekylære mekanismer for, for eksempel, den første celle afstamning beslutsomhed. Genetisk modifikation er en af ​​de mest almindeligt anvendte metoder til sikri...

Præimplantationsperioden fosterudviklingen kan opdeles i flere særskilte trin, fra en-celle stadiet til morula og blastocyst. Mange beviser tyder på, at polaritet og positionelle signaler spiller en rolle i tidlig celleskæbne bestemmelse i trophectoderm (TE) og den indre cellemasse (ICM). Arten af ​​de tidskoder, hvordan de transducerede til cellulære signaler, og de fysiologiske sammenhænge, ​​hvori sådanne signaler er i stand til at initiere cellelinie differentiering er ikke kendt. Disse differentierede celler derefter undergå specialisering, begyndt at tage på karakteristiske strukturer og funktioner, der er nødvendige for embryo ordentlig dannelse og vækst af moderkagen 1-3. Præimplantationsembryoner er særligt modtagelige for manipulation ved direkte injektion af modificerede genetiske materialer som siRNA eller target cDNA og således kan anvendes til undersøgelse af specifikke målmolekyler. Der er en voksende forståelse for, at genetiskanalyse af hvirveldyr embryoner er afgørende for at fremme vores forståelse af fosterudviklingen 4. Præcis indførelse af et vildtype-gen eller en mutant form i et embryo i rette sammenhæng at udrette GAIN- eller tab af genets funktion i høj grad har lettet studiet af udviklingsmæssigt regulerede gener. Tabs- og få af funktion via mikroinjektion af genetiske materialer i individuelle tidlige ikke-pattedyr og mammale embryoer er relativt enkel på grund af deres store størrelse og store modtagelighed 5. Mikroinjektion udføres typisk under anvendelse af ikke-virale vektorer. Særlig fordel er taget af den nemme at bruge ikke-virale vektorer i virale vektorer og transgener. En hurtig underskudsgivende eller gevinst af funktion ekspressionssystem i musefostre er blevet udviklet, der giver os mulighed for at dissekere og forstå genfunktioner i hvirveldyr embryologi 6,7. Fluorescerende mærkning af embryoner ville i høj grad lette udvælgelsen af ​​MICRoinjected eller genetisk manipulerede embryoer og samtidig yde et middel til indirekte at kvantificere ekspressionsniveauet af mikroinjiceres siRNA eller cDNA baseret på fluorescensintensitet 8. For at opnå dette mærkning, fluorescerende protein cDNA, GFP eller RFP, er co-injiceres som enten fusionskonstrukter eller hver for sig. Fluorescensintensiteten af ​​GFP eller RFP afslører omfanget af ekspression af siRNA eller fremmed DNA at sikre, at underskudsgivende eller få af funktion er blevet fuldført. Her præsenteres en mikromanipulering protokol for underskudsgivende og gain-of-funktion teknik, der tillod os at identificere neogenin som en receptor, som sender ekstracellulære signaler vigtige i begyndelsen af ​​celledifferentiering i præimplantationsperioden musefostre.

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			<td height="21" style="height:21px;width:496px;">Alexa fluor 488 labeled anti-rabbit antibody</td>
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			<td height="24" style="height:24px;width:496px;">all plasmid constructs&#160;</td>
			<td style="width:519px;">Prof. Wen Cheng Xiong at Georgia Health Sciences University (Agusta, GA).</td>
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			<td>the present studies were kindly provided by&#160;</td>
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			<td height="21" style="height:21px;width:496px;">grinder</td>
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			<td height="21" style="height:21px;width:496px;">Veriti&#174; 384-Well Thermal Cycler&#160;</td>
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loss- and gain-of-function approach to investigate early cell fate determinants in preimplantation mouse embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

Vi opdagede, at neogenin er forbigående udtrykkes i de tidlige udviklingsstadier af præimplantationsperioden musefostre, optræder så tidligt som ved 2-celle stadiet og varig indtil begyndelsen morula men bliver mangelfuld på det sene morula og blastocyst stadier (figur 2A). Desuden blev den rumlige fordeling af neogenin begrænset hovedsagelig til eksterne celler. Resultaterne af RT-PCR-analyse stemte overens med den tidlige og forbigående karakter neogenin ekspres...

Watch this Scientific Journal Video about Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos at JoVE.com

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Målet med denne protokol er at beskrive en underskudsgivende og få-funktion metode, der er anvendelig til at identificere neogenin som en scene-specifik receptor, som fører til trophectoderm og indre cellemasse differentiering i præimplantationsperioden musefostre.

Underskudsgivende og Gain-of-funktion tilgang til Undersøge Tidlig Cell Fate Determinanter i præimplantationsperioden Mouse Embryoner

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

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Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Metoder til Præcist Lokaliseret Overførsel af Celler eller DNA i Early implantation museembryoer

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Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

Kvantitativ analyse af Protein Expression til Studieinformationen Lineage Specification i Mouse præimplantationsperioden Embryoner

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

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Transkriptom profilering<em&gt; In-vivo</em&gt; Produceret Bovine præimplantationsembryoner Brug To farve Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

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