Name: structure-function studies in mouse embryonic stem cells using recombinase-mediated cassette exchange
Uploaded: 2017-04-27T15:00:00
Description: Watch this Scientific Journal Video about Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange at JoVE.com

Vi takker Jinke D&#39;Hont, Frederique Van Rockeghem, Natalie Farla, Kelly Lemeire, og Riet De RYCKE for deres utmerkede teknisk støtte. Vi takker også EEF Parthoens, Evelien Van Hamme og Amanda Goncalves fra Bioimaging Kjerne Facility av Betennelse Research Center for deres eksperthjelp. Vi erkjenner medlemmer av vår forskningsgruppe for verdifulle diskusjoner. Dette arbeidet ble støttet av den belgiske Science Policy (Belspo Interuniversity attraksjon polakker - Award IAP VII-07 DevRepair, https://devrepair.be), av dronning Elisabeth Medical Foundation, Belgia (GSKE 2008-2010; http: // www .fmre-gske.be), og ved å samordne forskning handlinger (GOA 01G01908) av Ghent universitet, Belgia (http://www.ugent.be/en/ghentuniv). SG er en postdoktor av Flandern forskningsmidler (FWO-V).

Alle forsøk på mus ble gjennomført i henhold til institusjonelle, nasjonale og europeiske dyre forskrifter. 1. Isolering av RMCE-kompatible KO mESCs <ol><li> Avle heterozygote KO-mus med RMCE-kompatibel mus, slik som ROSALUC mus 10 eller ROSA26-IPSC mus 21. Begge RMCE-kompatibel mus ble opprettholdt på en blandet 129 / C57BL6 / sveitsiske bakgrunn. MERK: Kryssing med heterozygote KO mus anbefales å overvinne embryonale dødelighet i homozygot KO mus. ...

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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=The+ground+state+of+embryonic+stem+cell+self-renewal.">The ground state of embryonic stem cell self-renewal.</a> Nature. 453, 519-523 (2008).</li><li>Chen, 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=Self-renewal+of+embryonic+stem+cells+by+a+small+molecule.">Self-renewal of embryonic stem cells by a small molecule.</a> Proc Natl Acad Sci U S A. 103, 17266-17271 (2006).</li><li>Pieters, T., 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=Efficient+and+User-Friendly+Pluripotin-based+Derivation+of+Mouse+Embryonic+Stem+Cells.">Efficient and User-Friendly Pluripotin-based Derivation of Mouse Embryonic Stem Cells.</a> Stem Cell Rev. 8, (2012).</li><li>Yang, 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=Pluripotin+combined+with+leukemia+inhibitory+factor+greatly+promotes+the+derivation+of+embryonic+stem+cell+lines+from+refractory+strains.">Pluripotin combined with leukemia inhibitory factor greatly promotes the derivation of embryonic stem cell lines from refractory strains.</a> Stem Cells. 27, 383-389 (2009).</li><li>Haenebalcke, 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=The+ROSA26-iPSC+Mouse:+A+Conditional,+Inducible,+and+Exchangeable+Resource+for+Studying+Cellular+(De)Differentiation.">The ROSA26-iPSC Mouse: A Conditional, Inducible, and Exchangeable Resource for Studying Cellular (De)Differentiation.</a> Cell Rep. 3, (2013).</li><li>Tucker, K. 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Vår Mesc isolasjonsmetoden er brukervennlig og krever ikke avanserte ferdigheter eller utstyr, som for eksempel mikro av blastocyster. Dermed er denne teknologien tilgjengelig for en stor andel av det vitenskapelige samfunn. Alle med grunnleggende cellekultur erfaring kan forplante ICM utvekster og etablere mESCs linjer. Men spyling og håndtering av blastocyster krever litt øvelse. En munn pipette brukes til å overføre blastocyster, og består av en mikropipette, en mikropipette holder, rør, og en sugemunnstykke <...

Det er anslått at pattedyr-genomer inneholder omtrent 20.000 protein-kodende gener. Alternativ spleising og posttranslasjonelle modifikasjoner ytterligere øke protein repertoaret. Proteiner har en modulær konstruksjon 1 og ofte inneholde flere interaksjons domener, noe som tillater deres rekruttering i forskjellige proteinkomplekser og deres deltagelse i flere cellulære prosesser 2. Et eksempel er den multifunksjonelle proteiner kjent p120ctn. p120ctn kodes for av Ctnnd1 gen og består av et stort sentralt armadillo gjenta domene flankert av en N-terminal og en C-terminal region. Armadillo domene av p120ctn binder seg til en høyt konservert juxtamembrane domene av klassiske cadheriner som er involvert i celle-celle-adhesjon, men det binder også til den transkripsjon repressoren Kaiso. Det N-terminale domenet av p120ctn samvirker med forskjellige kinaser, fosfataser, små RhoGTPases og mikrotubul-assosiert proteins tre. Det er interessant som et resultat av alternativ spleising, p120ctn isoformer kan genereres fra fire alternative startkodoner 4. p120ctn isoform 1A er den lengste, som det er oversatt fra den mest-5&#39; startkodon og inneholder full-lengde N-terminal segmentet. I p120ctn isoformer 3 og 4 er denne N-terminale segment slettes helt henholdsvis delvis og. Å forstå den nøyaktige rolle av proteiner (eller protein isoformene) og deres domenene i forskjellige cellulære funksjoner er fortsatt en utfordring. Gen-målsøking i mESCs gjør det mulig å studere funksjonen til et protein gjennom den genetiske delesjon av det tilsvarende genet og har bidratt mye til identifisering av utviklingsmessig viktige og sykdom-relevante gener og veier. Dette gjennombrudd i revers genetikk var et resultat av fremskrittene innen feltene Mesc isolasjon og gen-målsøking som følge av homolog rekombinasjon 5 </sopp&gt;. Homolog rekombinasjon er en prosess i hvilken DNA-fragmenter blir utvekslet mellom to lignende eller identiske nukleinsyrer grupper etter dobbelt-trådet (ds) DNA-brudd. Normalt er HR ineffektiv fordi dsDNA pauser er sjeldne. Nylig, kan effektiviteten av homologi-rettet gen-målsøking økes ved å bruke stedsspesifikke nuklease 6, 7, men dessverre disse er tilbøyelige til off-target effekter 8. En mer pålitelig teknikk for å muliggjøre gen-målsøking er RMCE, som er basert på setespesifikke rekombinasjon systemer som Cre / loxP eller FLPe / Frt. LoxP og Frt sekvens er funnet i bakteriofag P1 og Saccharomyces cerevisiae, henholdsvis, og består av 34 bp, inkludert et asymmetrisk 8 bp-sekvens som bestemmer orienteringen av området. På den annen side, kan orienteringen av, for eksempel, to loxP-seter innenfor et DNA strekning vil avgjøre hvorvidt den floxed DNA blir kuttet ut eller jegnversed ved Cre-formidlet rekombinasjon 9. Videre kan Cre også indusere en trans hvis to stedene ligger på forskjellige kromosomer. RMCE utnyttet heterospecific rekombinasjonsseter som ikke kryssreagerer og som er innebygd i et genom-locus. I nærvær av en donor plasmid som inneholder et DNA-fragment flankert av de samme heterospecific steder, vil den rekombinasen sette inn dette DNA-fragment i RMCE-kompatible genomiske lokuset på grunn av dobbel-samtidig translokasjon (figur 1). Her kan bare korrekt RMCE rettede kloner gjengi medikamentresistens, takket være en promoter på den innkommende vektoren som gjenoppretter en &quot;fanget&quot; promoter-mindre neomycin resistensgenet (neo R) som er tilstede i R26 genomet av dokkings-celler (Figur 1) 10, 11. Dette resulterer i en meget høy effektivitet målretting, ofte nær 100% 11 &lt;/ sup&gt; 12. Som konklusjon, er RMCE basert målretting svært effektiv og kan anvendes for struktur-funksjoner studier; Men det krever en pre-utviklet genomisk locus. <img alt="Figur 1" src="/files/ftp_upload/55575/55575fig1.jpg" /> Figur 1. Skjematisk representasjon av RMCE-mediert målretting. RMCE tillater utveksling av DNA-segmenter fra en innkommende målsøkende vektor til en definert genomisk locus hvis begge huse to heterospecific FRT steder (vist med hvite og røde trekanter). I tillegg inneholder det konstruerte genomiske lokuset en promoterfrie og avkortet neomycin-resistens (Neo R) genet. Ved å tilveiebringe en promoter og startkodon i den innkommende DNA-fragment, kun korrekt rekombinasjonshendelser gjenopprette neomycinresistens, som resulterer i høye målretting effektivitet. <a href="http://ecsource.jove.com/files/ftp_upload/55575/55575fig1large.jpg" target="_blank">Klikk her for å se en større versjon av t</a>hans skikkelse. Genome engineering i mESCs tillater generering av RMCE-kompatibel mus. I 1981 ble to grupper lyktes å pluripotente celler fra den indre cellemasse (ICM) av blastocyster, og i å opprettholde dem i kultur 13, 14. mESCs er i stand til selvfornyelse og differensiering i alle typer av embryonale og voksne celler, inkludert bakterie-cellelinjen. Derfor, gen-målsøking i mESCs muliggjør revers-genetiske studier gjennom utviklingen av konstitutive eller betingede (ved bruk av Cre / loxP-system) KO-mus. Imidlertid er den klassiske metoden for å isolere muse-ES-celler er svært ineffektiv. Flere store forbedringer har sterkt øket suksessraten for utledning av Mesc linjer, inkludert bruk av et definert serum erstatning (SR) medium 15, alternerende mellom Mesc medium inneholdende SR og føtalt bovint serum (FBS) 16, og bruken av farmakologiskelogiske forbindelser slik som pluripotin eller 2i 17. Pluripotin, en liten syntetisk molekyl, tillater forplantning av mESCs i en udifferensiert tilstand i fravær av leukemi-inhiberende faktor (LIF) og muse-embryonale fibroblaster (MEFs) 18. Endelig har det vist seg at mESCs kan isoleres med en meget høy virkningsgrad (tilnærmet 100%) ved en SR / FBS-medium alternering protokoll er kombinert med LIF og pluripotin 19, 20. Disse protokollene muliggjøre effektiv isolering av RMCE-kompatible KO mESCs som senere kan brukes for struktur-funksjonsstudier. Dette dokumentet beskriver en metode som gjør det mulig å identifisere de viktigste domener eller ester i et protein som er ansvarlig for spesifikke cellulære prosesser. For dette formål er en rørledning av avansert teknologi som muliggjør effektiv Mesc isolasjon, målsøkende vektor montasje, og Mesc målretting var skaped. Som sådanne store paneler med proteinisoformer, domene mutanter, og nedstrøms effektorer kan innføres i KO mESCs og kan evalueres for deres evne til å redde in vitro KO fenotype.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">ROSALUC Mice</td>
			<td style="width:143px;">made in house</td>
			<td style="width:107px;"></td>
			<td style="width:245px;">frozen sperm available upon request</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">R26-iPSC mice</td>
			<td style="width:143px;">made in house</td>
			<td style="width:107px;"></td>
			<td style="width:245px;">frozen sperm available upon request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Vessel probe</td>
			<td style="width:143px;">Fine Science Tools</td>
			<td>10160-13</td>
			<td style="width:245px;">to check for copulation plugs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">M2 medium</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:107px;">M7167</td>
			<td style="width:245px;">make aliquots and store at -20&#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">Fine forceps (Dumont #5 Standard tip Student forceps)</td>
			<td style="width:143px;">Fine Science Tools</td>
			<td style="width:107px;">11251-10</td>
			<td style="width:245px;">spray with 70% EtOH before use (do not autoclave)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">23G needles</td>
			<td style="width:143px;">Fine-ject</td>
			<td style="width:107px;">8697</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">1-ml syringes</td>
			<td style="width:143px;">Soft-ject</td>
			<td style="width:107px;">6680</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">60-mm bacterial grade plates (for flushing)&#160;</td>
			<td style="width:143px;">Gosselin</td>
			<td style="width:107px;">BB60-01</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Mouth pipette</td>
			<td style="width:143px;">made in house</td>
			<td style="width:107px;"></td>
			<td style="width:245px;">see discussion</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">Mouse embryonic fibroblasts (MEFs, TgN (DR4)1 Jae strain)</td>
			<td style="width:143px;">ATTC</td>
			<td style="width:107px;">SCRC-1045</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">TgN (DR4)1 Jae mice</td>
			<td style="width:143px;">The Jackson Laboratory</td>
			<td style="width:107px;">3208</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Mitomycin C&#160;</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:107px;">M0503</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">Phosphate buffered saline (PBS) without calcium or magnesium</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">14190-094</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:272px;">Tg(DR4)1Jae/J mice</td>
			<td style="width:143px;">JAX</td>
			<td style="width:107px;">3208</td>
			<td style="width:245px;">mice that contain four drug-selectable genes and DR4 MEFS confers resistance to neomycin, puromycin, hygromycin and 6-thioguanine</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">0.1% Gelatin</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:107px;">G1393</td>
			<td style="width:245px;">Dissolve 0.5 g in 500 ml distilled water, autoclave and store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Trypsin (0.25%)&#160;</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">25200-056</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">2 &#956;M pluripotin</td>
			<td style="width:143px;">Cayman Chemical</td>
			<td style="width:107px;">10009557</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:272px;">pRMCE-DV1</td>
			<td style="width:143px;">BCCM/LMBP collection&#160;</td>
			<td style="width:107px;">LMBP 08870</td>
			<td style="width:245px;">public available from the BCCM/LMBP collection (http://bccm.belspo.be)&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:272px;">cre-excised pRMCE-DV1</td>
			<td style="width:143px;">BCCM/LMBP collection&#160;</td>
			<td style="width:107px;">LMBP 08195</td>
			<td style="width:245px;">public available from the BCCM/LMBP collection (http://bccm.belspo.be)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">pCAG-FlpE-IRES-Puro-pA&#160;</td>
			<td style="width:143px;">Addgene</td>
			<td style="width:107px;">20733</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">heat-shock competent DH5&#945; bacteria&#160;</td>
			<td style="width:143px;">made in house</td>
			<td style="width:107px;"></td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">Gateway pDONR221 vector</td>
			<td style="width:143px;">Thermo Fisher</td>
			<td style="width:107px;">12536-017</td>
			<td style="width:245px;">contains kanamycin-resistance gene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BP clonase II mix</td>
			<td style="width:143px;">Thermo Fisher</td>
			<td style="width:107px;">11789-020</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">LR clonase II mix</td>
			<td style="width:143px;">Thermo Fisher</td>
			<td style="width:107px;">11791-020&#160;</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Luria Broth (LB)&#160;</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Ampicillin&#160;</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Applied Biosystems 3730XL DNA Analyzer</td>
			<td style="width:143px;">Thermo Fisher</td>
			<td style="width:107px;">3730XL</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">G418</td>
			<td style="width:143px;">Thermo Fisher</td>
			<td style="width:107px;">11811-023</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">Lipofectamine 2000 transfection reagent</td>
			<td style="width:143px;">Thermo Fisher</td>
			<td style="width:107px;">11668027</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Lipofectamine LTX transfection reagent</td>
			<td style="width:143px;">Thermo Fisher</td>
			<td style="width:107px;">15338100</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Effectene transfection reagent</td>
			<td style="width:143px;">Qiagen</td>
			<td style="width:107px;">301425</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">GATEWAY pENTR 1A vector</td>
			<td style="width:143px;">Thermo Fisher</td>
			<td style="width:107px;">A10462</td>
			<td>recombination-compatible vector</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">mouse monoclonal anti-p120ctn antibody</td>
			<td style="width:143px;">BD Transduction Laboratories</td>
			<td style="width:107px;">610134</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">mouse monoclonal anti-Ecadherin antibody</td>
			<td style="width:143px;">BD Transduction Laboratories</td>
			<td style="width:107px;">610181</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">General equipment:</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">Sterile dissection tools</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td style="width:245px;">fine scissors and forceps for dissecting the uterus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Sterile pipettes: 5 ml, 10 ml and 25 ml</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">15-ml and 50-ml conical centrifuge tubes</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:272px;">96-well culture plates V-shaped bottom and flat bottom)&#160;</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">Culture dishes: 24 wells, 12 wells and 6 wells</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">Multichannel pipettes (to pipette 30, 50, 100 and 200 &#956;l)&#160;</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Sterile multichannel reservoirs</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Access to a laminar air flow</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:272px;">Access to an incubator at 37&#176;C with 5% CO2</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Access to an inverted microscope</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Access to a bench-top centrifuge</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">Access to a stereo microscope with transmitted-light&#160;</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Culture media:</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">MEF Medium:</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td style="width:245px;">stored at 4&#176;C; warm 30 min at 37&#176;C before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">&#160;Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">41965-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">10% fetal bovine serum (FBS)</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:107px;">F-7524</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">L-glutamine (2 mM)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">25030-024&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Sodium pyruvate&#160; (0.4 mM)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">11360-039&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">penicillin (100 U/ml)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">15140-122&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">streptomycin (100 &#181;g/ml)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">15140-122&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">SR-based mESC medium:</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td style="width:245px;">stored at 4&#176;C; warm 30 min at 37&#176;C before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">DMEM/F12</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">31330-038</td>
			<td style="width:245px;">mixed in a 1:1 ratio</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">15% knock-out serum replacement (SR )</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">10828&#8211;028</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">L-glutamine (2 mM)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">25030-024&#160;</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">0.1 mM non-essential amino acids&#160;</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">11140-050</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">penicillin (100 U/ml)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">15140-122&#160;</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">streptomycin (100 &#181;g/ml)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">15140-122&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">&#946;-mercaptoethanol (0.1 mM)&#160;</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:107px;">M 3148&#160;</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">2,000 U/ml recombinant mouse LIF&#160;</td>
			<td style="width:143px;">(IRC/VIB Protein Service facility)</td>
			<td style="width:107px;"></td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">FBS-based mESC medium (similar to SR-based mESC medium):</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td style="width:245px;">stored at 4&#176;C; warm 30 min at 37&#176;C before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Knockout DMEM</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">10829-018&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">15% FBS</td>
			<td style="width:143px;">Hyclone</td>
			<td style="width:107px;">SH30070.03E</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Differention Medium</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td style="width:245px;">stored at 4&#176;C; warm 30 min at 37&#176;C before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">21980-032&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">15% FBS</td>
			<td style="width:143px;">Hyclone</td>
			<td style="width:107px;">SH30070.03E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">5% CD Hybridoma Medium(1x) liquid&#160;&#160;</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">11279-023&#160;</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">&#160;2 mM L-glutamine</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">25030-024&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">0.4 mM 1-thioglycerol</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:107px;">M-6145</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">50 &#956;g/ml ascorbic acid</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:107px;">A-4544&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">penicillin (100 U/ml)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">15140-122&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">streptomycin (100 &#181;g/ml)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:107px;">15140-122&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2i</td>
			<td style="width:143px;"></td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">1 &#956;M Erk inhibitor PD0325901&#160;</td>
			<td style="width:143px;">Axon Medchem</td>
			<td style="width:107px;">Axon 1408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">3 &#956;M Gsk3 inhibitor CHIR99021</td>
			<td style="width:143px;">Axon Medchem</td>
			<td style="width:107px;">Axon 1386</td>
			<td></td>
		</tr>
	</tbody>
</table>

structure-function studies in mouse embryonic stem cells using recombinase-mediated cassette exchange

Gene teknikk i museembryoer eller embryonale stamceller (mESCs) gjør det mulig å studere funksjonen av et gitt protein. Proteiner er arbeidshester av cellen og ofte bestå av flere funksjonelle domener, som kan påvirkes av post-translasjonelle modifikasjoner. Uttømming av hele proteinet i kondisjon eller konstitutiv knock-out (KO) mus som ikke tar hensyn til denne funksjonell diversitet og regulering. En Mesc linje og en avledet musemodell, hvor en festestedet for FLPe rekombinasjon-mediert kassett utveksling (RMCE) ble innført i ROSA26 (R26) locus, ble tidligere rapportert. Her rapporterer vi på en struktur-funksjon tilnærming som gjør det mulig for molekylær disseksjon av de ulike funksjonene i en multidomain protein. For å oppnå dette, må RMCE-kompatible mus krysses med KO mus og deretter RMCE-kompatible KO mESCs må isoleres. Deretter kan et panel av antatte rednings konstruksjoner innføres i R26 locus via RMCE targeting. Kandidat-rednings cDNA kan enkelt settes inn mellom RMCE områder av den målsøkende vektor ved hjelp av rekombinasjon kloning. Deretter blir KO mESCs transfektert med den målsøkende vektor i kombinasjon med et FLPe rekombinase ekspresjonsplasmid. RMCE reaktiverer den promoter-mindre neomycin-resistensgenet i ROSA26 tilkoblings områder og muliggjør valg av den riktige målretting arrangementet. På denne måten blir høy målretting virkningsgrader nær 100% oppnådd, som tillater innføring av flere mulige rednings konstruksjoner i en semi-høy gjennomstrømning måte. Endelig kan et mangfold av R26-drevne rednings konstruksjoner bli testet for deres evne til å redde den fenotype som ble observert i foreldre KO mESCs. Vi presenterer et bevis-for-prinsipp struktur-funksjon studie med p120 catenin (p120ctn) KO mESCs ved hjelp endoderm differensiering i embryoidlegemene (EBS) som fenotypiske avlesning. Denne tilnærmingen muliggjør identifikasjon av viktige områder, antatte nedstrøms trasé, og sykdomsrelevant punktmutasjoner som ligger til grunn KO fenotyper for et gitt protein.

Fremgangsmåten for å isolere RMCE-kompatibel KO Mesc linjer er vist i figur 2. To påfølgende ynglinger er nødvendig for å oppnå RMCE-kompatibel KO blastocyster. Først blir heterozygote KO mus krysset med RMCE-kompatible mus for å oppnå RMCE-kompatibel, heterozygot KO-mus. Disse mus ble så brukt for tidsbestemt parringer med andre heterozygote KO-mus for å oppnå 3,5-DPC, RMCE-kompatibel, homozygote KO-blastocyster. Sjansen for å få en slik embryo er en av ?...

Watch this Scientific Journal Video about Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange at JoVE.com

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Proteiner inneholder ofte flere domener som kan utøve ulike cellulære funksjoner. Gene knock-outs (KO) betrakter ikke dette funksjonelle mangfold. Her rapporterer vi en rekombinasjon-mediert kassett utveksling (RMCE) -basert struktur-funksjonsmetoden i KO-embryonale stamceller som gjør det mulig for den molekylære disseksjon av forskjellige funksjonelle domener eller varianter av et protein.

Struktur-funksjon studier i mus embryonale stamceller Bruke recombinase-mediert Cassette Veksling

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

The overall goal of this structure-function method is to dissect at the molecular level the role of different protein domains, or disease-relevance mutations, in naive or differentiated mouse embryonic stem cells. This method can help to reveal how different residues or domains of a protein can contribute to its function in a given cellular context. The main advantage of this technique is that it allows very efficient targeting of rescue constructs to the genome of knock out embryonic stem cells, or ES cells, and the investigation of their role in different cell types.To achieve this, we combine three highly efficient technologies. These include improved mouse embryonic stem cell isolation, recombination-based targeting factor assembly, and ES cell targeting via recombination-mediated cassette exchange, or RMC. Demonstrating some of the procedures will be Jinke D'Hont, a technician from my laboratory.Recombination-mediated cassette exchange, or RMCE, allows the interchange of DNA fragments between a vector and a genomic locus. RMCE takes advantage of hetero-specific recombination sites that do not cross-react and that are embedded in a genomic locus. In the presence of a donor plasmid that contains a DNA fragment flanked by the same hetero-specific sites, the recombinase will insert this DNA fragment into the RMCE-compatible genomic locus because of double simultaneous translocation.Only upon correct recombination, the trapped promoterless Neomycin-resistance gene in the Rosa 26 docking site, will be restored via a PGK promoter in the incoming targeting vector, which renders drug-resistance. This Neomycin resistance trap system results in a very high targeting efficiency, often close to 100%The day before blastocyst isolation, add 0.1%gelatin, to 12 well-cultured plates. And incubate them at 37 degrees Celsius for five minutes.Aspirate the gelatin solution. Then seed one quarter of a vial of P2 Mouse Embryonic Fibroblasts, or MEFs, into MEF medium, and divide over the 12-well plate. Incubate the 12-well plates of MEFs in two milliliters of MEF medium at 37 degrees Celsius.And grow the cells to a confluent monolayer. Then add 10 micrograms per milliliter of Mitomycin seed to the wells. And incubate the cultures for three hours at 37 degrees Celsius, to inactivate the cells.After selecting heterozygous knock out mice containing an RMCE cassette in the Rosa 26 locus according to the text protocol, set up matings to breed RMCE-compatible heterozygous knock out mice, with heterozygous knock out mice. The following morning, check for copulation plugs by lifting the female by the base of the tale, and examine the vaginal opening for a whitish mass. If the plug is difficult to see, with an angled probe, slightly spread the lips of the vulva, then separate the plugged females from their males.To collect blastocysts at 3.5 days post-coitus, or DPC. After euthanizing the pregnant females, make a mid-ventral incision, and use fine scissors and forceps to dissect the uterus and oviduct. Next, bend a 26-gauge needle into a 45-degree angle attached to a 1 milliliter syringe filled with M2 medium.And insert the needle into the end of the uterus that is closest to the oviduct. Use fine forceps to hold the needle in place while pushing the plunger to flush the blastocysts from the uterus into the lid of a 10 centimeter dish. The uterus will swell, if the flushing is successful.Use a mouth pipette to collect all the embryos in a drop of M2 medium, and wash the embryos twice in a drop of M2 medium. Immediately after washing the embryos, use PBS, to wash the mitomycin-C inactivated cells twice. Then using a mouth pipette, plate each blastocyst onto a separate well of the mitomycin-C treated MEFs, in SRES cell medium, supplemented with pluripotent or 2I.Incubate the cultures at 37 degrees Celsius, and 5%carbon dioxide. Refresh the SREF cell medium every two to three days. Examine each blastocyst under a stereo microscope at 4X magnification, and check for hatching and detachment to the MEF layer.After 10 to 12 days of culture, under a stereo microscope, use a P10 pipette with disposable tips, to pick individual icium outgrowths. Transfer each outgrowth into approximately 10 microliters of medium to a V-shaped 96-well plate, containing 30 microliters per well of PBS. Using a multi-channel pipette, add 50 microliters of 0.25%trypsin to each well.And incubate the plate at 37 degrees Celsius and 5%carbon dioxide for three minutes. Add 100 microliters of pre-incubated FPS-containing ES cell medium, and dissociate the icium outgrowths into single cells by pipetting 10 to 15 times. Then transfer the dissociated cells to mitomycin-C treated 96 well MEF plates.The following day, change the SR-based medium. Expand the ES cells in a similar fashion from 24 to 6-well plates. After identifying rescued CDNA vectors according to the text protocol, prepare a 10 microliter LR reaction using 100 nanograms of rescue CDNA vector, 150 nanograms of pre-excised RMCEDV1 vector, and two microliters of recombinase mix, containing a phage-encoded integrase, an excisionase, and a bacterial integration host factor.Incubate the reaction at 25 degrees Celsius for two hours. To transform the recombined vectors, add 5 microliters of each mixture, to 40 microliters of heat shock competent E.Coli bacteria, in a 2 milliliter skirted screw cap tube. And incubate the sample on ice for 20 minutes.Then incubate the cells at 37 degrees Celsius for five minutes. Add one milliliter of LB medium to the tube, and incubate the cells at 37 degrees Celsius for one hour. Plate 50 microliters on agar plates with ampicillin.And grow at 37 degrees Celsius overnight. Identify colonies with the correct targeting vector by using a P200 tip to randomly pick five colonies. Transfer the tip to a glass test tube containing two to five milliliters of LB medium, and grow overnight at 37 degrees Celsius.After extracting the DNA from each colony, validate the samples by digesting 0.5 to two micrograms of DNA, and separate the samples on a 1%agarose gel. Start a culture of RMCE compatible knock out ES cells, and passage them at least twice on MEFs, in FBS-based ES cell medium. Then split the ES cells on a gelatinized six-well plate.The following day, use 1.5 milliliters of FBS-based ES cell medium to refresh the ES cells that are about 50%confluent. Combine one microgram of pre-excised RMCEDV1 targeting vector, containing rescue CDNA, and one microgram of FLIPe expression plasmid, in 250 microliters of pure DMEM medium. Add seven microliters of lipofectin-based transvection reagent to 250 microliters of pure DMEM medium.And incubate the solution for five minutes at room temperature. Combine the DNA and lipofectin solutions. And incubate the mixture at room temperature for 20 minutes.Then pipette the transvection mixture onto the refreshed ES cells, and gently swirl. One day after transvection, split all ES cells from the tube to a 10-centimeter culture dish, with a confluent layer of DR4 MEFS, and 10 milliliters of FPS-based ES cell medium. Two days after transvection, select ES cell clones with the correct FLIP-e mediated cassette exchange, by adding G418 to the medium.As shown here, massive killing of non-targeted colonies should be visible after three to five days. Colonies should appear after seven to 10 days. Pick these colonies, then expand them, and verify the clones as demonstrated earlier in this video.To differentiate ES cells into embryoid bodies, or EBs, after culturing knock out ES cells with Rosa 26-driven rescue constructs according to the text protocol, allow EBs to form in the non-adherent dishes for 30 days. Refresh the medium every two to three days by transferring the EB suspension to a 50 milliliter tube. And let the EBs settle by gravity.Remove the supernatant, add fresh medium, and transfer the EB suspension to a bacterial-grade dish. Analyze the ES cells and EBs by immunofluorescence and TEM, according to the text protocol. Using the structure function method, five pre-excised RMCEDV1 targeting vectors were generated with an efficiency of 100%and these rescue constructs were targeted via RMCE, to P120 catenin knock out ES cells, with an efficiency of 97%Cystic EB morphology can be used as a phenotypic readout to screen different rescue constructs.As proof of concept, R26-driven P120 catenin isoform 1A rescued the p120 catenin knock out phenotype. To test whether E-cadherin independent membrane anchoring of P120 catenin enabled csytic EB formation, the k-ras membrane targeting motif, CAAX, was fused to the carboxy terminus of P120 catenin, and introduced via RMCE, to P120 catenin knock out ES cells, before EBs were made from them. However, this construct serves a dominant negative that does not allow binding and stabilization of E-cadherin.And as a consequence, does not rescue the p120 catenin knock out phenotype. Epithelial-mesenchymal transition, or EMT, is an essential developmental process that also occurs during ES cell differentiation. When expressed in p120 catenin knock out ES cells, the EMT inducers, ZEB1, ZEB2, or snail that can directly repress various epithelial marker genes like E-cadherin, failed to restore cystic EB formation.Once mastered, RMCE compatible ES cells can be isolated within one month. RMCE compatible targeting vectors can be generated within one week, and targeted rescue of ES cells can be achieved within one month using this technique, if it is performed properly. After watching this video, you should have a good understanding of how to use RMCE to perform structure function studies in naive or differentiated embryonic stem cells.

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