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

Wij danken Jinke D&#39;Hont, Frederique Van Rockeghem, Natalie Farla, Kelly Lemeire en Riet De Rycke voor hun uitstekende technische ondersteuning. We danken ook Eef Parthoens, Evelien Van Hamme, en Amanda Goncalves uit de bio-imaging Core Facility van de Ontsteking Onderzoekscentrum voor hun deskundige hulp. Wij erkennen de leden van onze onderzoeksgroep voor waardevolle discussies. Dit werk werd ondersteund door het Federaal Wetenschapsbeleid (Belspo Interuniversitaire attractiepolen - Award IAP VII-07 DevRepair; https://devrepair.be), door de Geneeskundige Stichting Koningin Elisabeth, België (GSKE 2008-2010; http: // www .fmre-gske.be), en door de gecoördineerde Research Actions (GOA 01G01908) van de Universiteit Gent, België (http://www.ugent.be/en/ghentuniv). SG is een postdoctoraal onderzoeker van het Flanders Research Funds (FWO-V).

Alle experimenten met muizen werden uitgevoerd volgens institutioneel, nationaal en Europees dierenpaspoort regelgeving. 1. Isolatie van RMCE-compatibele KO mESCs <ol><li> Ras heterozygote KO muizen met RMCE-compatibele muizen, zoals ROSALUC muizen 10 of ROSA26-iPSC 21 muizen. Beide RMCE-compatibele muizen werden gehouden op een gemengd 129 / C57BL6 / Swiss achtergrond. LET OP: De kruising met heterozygote KO muizen wordt geadviseerd om embryonale dodelijk...

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Onze mES isolatie methode is gebruiksvriendelijk en vereist geen geavanceerde vaardigheden of apparatuur, zoals microchirurgie van blastocysten. Zo, deze technologie is toegankelijk voor een groot deel van de wetenschappelijke gemeenschap. Iedereen met basic celkweek ervaring kan voortplanten ICM uitwassen en stellen mESCs lijnen. Echter, de spoelen en de behandeling van blastocysten enige oefening vereist. Een mond pipet wordt gebruikt om blastocysten overdracht en bestaat uit een micropipet, een micropipet houder, sla...

Geschat wordt dat zoogdieren genomen bevatten ongeveer 20.000 eiwitcoderende genen. Alternatief splitsen en posttranslationele modificaties verder te verhogen het eiwit repertoire. Eiwitten een modulaire constructie 1 en bevatten vaak meerdere interactie-domeinen, waardoor hun rekrutering in verschillende eiwitcomplexen en hun deelname aan verschillende celprocessen 2 mogelijk. Een voorbeeld is de multifunctionele eiwit genaamd p120ctn. p120ctn wordt gecodeerd door het gen Ctnnd1 en bestaat uit een groot centraal armadillo herhalende domein geflankeerd door een N-terminale en C-terminale gebied. De armadillo domein van p120ctn bindt aan een sterk geconserveerd juxtamembraandomein klassieke cadherinen, die betrokken zijn bij cel-cel adhesie, maar bindt ook aan de transcriptionele repressor Kaiso. Het N-terminale domein van p120ctn interageert met andere kinasen, fosfatasen, kleine RhoGTPases en microtubule geassocieerde pProteins 3. Interessant is dat als gevolg van alternatieve splicing, p120ctn isovormen kunnen worden gegenereerd uit vier alternatieve startcodons 4. p120ctn isovorm 1A is het langst, zoals vertaald vanaf de meest 5&#39; startcodon en bevat de volledige lengte N-eindstandige segment. In p120ctn isovormen 3 en 4, wordt dit N-eindstandige segment gedeeltelijk en volledig respectievelijk verwijderd. Inzicht in de precieze rol van eiwitten (of eiwit isovormen) en hun domeinen in verschillende cellulaire functies blijft een uitdaging. Gen-targeting in mESCs maakt het bestuderen van de functie van een proteïne door genetische deletie van het overeenkomstige gen en heeft een grote bijdrage tot de identificatie van ontwikkelings belangrijk en ziekte-relevante genen en pathways. Deze doorbraak in reverse genetics was het gevolg van de ontwikkelingen op het gebied van mES isolatie en gene targeting als gevolg van homologe recombinatie 5 </sup&gt;. Homologe recombinatie is een werkwijze waarbij DNA-fragmenten worden uitgewisseld tussen twee gelijkaardige of identieke nucle- resten na dubbelstrengs (ds) DNA-breuken. Normaal gesproken, HR is inefficiënt omdat dsDNA pauzes zijn zeldzaam. Onlangs zou de efficiëntie van homologie gerichte gerichte mutagenese worden verhoogd middels plaatsspecifieke nucleasen 6, 7, maar helaas zijn deze gevoelig voor off-doeleffecten 8. Een meer betrouwbare techniek voor gen targeting inschakelen is RMCE, die gebaseerd is op plaatsspecifieke recombinatie systeem zoals Cre / loxP of FLPe / Frt. LoxP en Frt sequentie in bacteriofaag P1 en Saccharomyces cerevisiae, respectievelijk, en bestaan uit 34 bp, waaronder een asymmetrische 8 bp sequentie die de oriëntatie van de site bepaalt. Anderzijds de oriëntatie van, bijvoorbeeld twee loxP plaatsen in de DNA strook bepaalt of de floxed DNA wordt uitgesneden of inversed upon Cre gemedieerde recombinatie 9. Bovendien kan Cre ook translocatie induceert wanneer twee plaatsen zich op verschillende chromosomen. RMCE maakt hiervan heterospecifiek recombinatie locaties die niet kruisreageren en die zijn ingebed in een genomische locus. In aanwezigheid van een donor plasmide dat een DNA-fragment geflankeerd door dezelfde heterospecifiek plaatsen bevat, zal de recombinase dit DNA-fragment in te voegen in de RMCE-compatibele genomische locus verdubbelen als gevolg van gelijktijdige translocatie (figuur 1). Hier kunnen alleen correct RMCE gerichte klonen render geneesmiddelresistentie door een promotor van de inkomende vector die herstelt een &quot;gevangen&quot; promoter-less neomycine resistentiegen (neo R) in de R26 genoom van het docking cellen (figuur 1) 10, 11. Dit resulteert in een zeer hoge targeting efficiency, vaak in de buurt van 100% 11, &lt;/ sup&gt; 12. Concluderend-RMCE gebaseerde targeting is zeer efficiënt en kan worden gebruikt voor structuur-functie studies; echter, het vereist een prefab genomische locus. <img alt="Figuur 1" src="/files/ftp_upload/55575/55575fig1.jpg" /> Figuur 1. Schematische weergave van RMCE-gemedieerde targeting. RMCE maakt de uitwisseling van DNA-segmenten uit een inkomend richtende vector op een bepaalde genomische locus als zowel haven heterospecifiek twee FRT-plaatsen (aangegeven door witte en rode driehoekjes). Bovendien, de gemanipuleerde genome locus bevat een promoter en afgeknot neomycine-resistentie (Neo R) gen. Door een promoter en startcodon in het inkomende DNA-fragment, enige juiste recombinatiegebeurtenissen herstellen neomycine resistentie, wat resulteert in hoge rendementen targeting. <a href="http://ecsource.jove.com/files/ftp_upload/55575/55575fig1large.jpg" target="_blank">Klik hier om een grotere versie van t bekijken</a>zijn figuur. Genome Engineering in mESCs maakt het mogelijk om het genereren van RMCE-compatibele muizen. In 1981, twee slaagden in het vastleggen van pluripotente cellen van de binnenste celmassa (ICM) van blastocysten en het handhaven in kweek 13, 14. mESCs in staat zijn zelfvernieuwing en differentiatie tot alle embryonale en volwassen cellen, inclusief de geslachtscellen-cellijn. Daarom gen-targeting in mESCs maakt reverse genetische studies door de ontwikkeling van constitutieve of voorwaardelijke (via het Cre / loxP systeem) KO muizen. Echter, de klassieke manier om de muis ES-cellen te isoleren is zeer inefficiënt. Aantal belangrijke verbeteringen sterk verhoogd succes voor het afleiden mES lijnen, inclusief het gebruik van een bepaald serum-vervanging (SR) -medium 15, afwisselend mES medium met SR en foetaal runderserum (FBS) 16 en het gebruik farmacologische verbindingen zoals pluripotin of 17 2i. Pluripotin, een klein synthetisch molecuul, maakt de voortplanting van mESCs in een ongedifferentieerde toestand bij afwezigheid van leukemie remmende factor (LIF) en muis embryonale fibroblasten (MEF) 18. Tenslotte is aangetoond dat mESCs kan worden geïsoleerd met een zeer hoog rendement (bijna 100%) wanneer een SR / FBS medium afwisseling protocol wordt gecombineerd met LIF en pluripotin 19, 20. Deze protocollen maken de efficiënte isolatie van RMCE-compatibele KO mESCs die vervolgens kunnen worden gebruikt voor structuur-functie studies. Dit artikel beschrijft een werkwijze die het mogelijk maakt om de belangrijkste domeinen of resten te identificeren in een eiwit dat verantwoordelijk is voor specifieke cellulaire processen. Te dien einde, een pijpleiding van geavanceerde technologieën die het mogelijk maken een efficiënte mES isolatie, targeting vector assemblage en mES targeting was creërend. Als zodanig grote panelen met eiwit isovormen, domain mutanten en downstream effectoren in KO mESCs kan worden ingevoerd en kan worden geëvalueerd op hun vermogen om de in vitro KO fenotype te redden.

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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>
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			<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

Genmanipulatie in muizenembryo&#39;s of embryonale stamcellen (mESCs) maakt voor de studie van de functie van een bepaald eiwit. Eiwitten zijn de werkpaarden van de cel en vaak uit meerdere functionele domeinen, die kan worden beïnvloed door posttranslationele modificaties. De uitputting van het gehele eiwit in conditionele of constitutief knock-out (KO) muizen geen rekening houden met deze functionele diversiteit en regelgeving. Een mES lijn en een afgeleide muismodel, waarbij een docking site voor FLPe-recombinatie gemedieerde cassette uitwisseling (RMCE) binnen de ROSA26 (R26) locus was ingebracht, werd eerder beschreven. Hier doen we verslag van een structuur-functie benadering die het mogelijk maakt voor de moleculaire dissectie van de verschillende functionaliteiten van een multidomein eiwit. Daartoe moet RMCE compatibele muizen worden gekruist met KO muizen en RMCE-compatibele KO mESCs moeten worden geïsoleerd. Vervolgens kan een panel van vermeende redding constructen worden geïntroduceerd in de R26 locus via RMCE targeting. De kandidaat rescue cDNAs kunnen gemakkelijk ingebracht tussen RMCE plaatsen van de richtende vector door middel van recombinatie klonering. Vervolgens worden KO mESCs getransfecteerd met het richtende vector in combinatie met een FLPe recombinase expressieplasmide. RMCE reactiveert het promoter-loze neomycine-resistentiegen in het ROSA26 aanlegplaatsen en maakt de keuze van de juiste targeting. Daardoor worden hoge targeting efficiëntie bijna 100% verkregen, waardoor het inbrengen van meerdere mogelijke redding constructen in een semi-high throughput manier. Tenslotte kan een veelvoud van R26-aangedreven rescue constructen worden getest op hun vermogen om het fenotype dat waargenomen ouderlijke KO mESCs redden. We presenteren een proof-of-principle structuur-functie studie p120 catenine (p120ctn) KO mESCs behulp endoderm differentiatie embryo lichamen (EB&#39;s) als fenotypische uitlezing. Deze benadering maakt de identificatie van belangrijke domeinen vermoedelijke stroomafwaartse routes en ziekte-relevante gegevensmutaties die KO fenotypes voor een bepaald eiwit ten grondslag liggen.

De procedure voor RMCE-compatibele KO mES lijnen te isoleren is weergegeven in figuur 2. Twee opeenvolgende dekkingen zijn verplicht om RMCE-compatibele KO blastocysten te verkrijgen. Eerst worden heterozygote KO muizen gekruist met RMCE-compatibele muizen om RMCE-compatibele, heterozygote KO muizen te verkrijgen. Deze muizen worden vervolgens gebruikt voor getimede paringen met andere heterozygote KO muizen 3,5 dpc, RMCE-compatibele homozygote KO blastocysten verkregen....

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

Eiwitten vaak meerdere domeinen die verschillende cellulaire functies kunnen uitoefenen. Gene knock-outs (KO) beschouw dit niet als functionele diversiteit. Hier melden wij een recombinatie-gemedieerde cassette uitwisseling (RMCE) gebaseerde structuur-functie benadering KO embryonale stamcellen waarmee de moleculaire dissectie van verschillende functionele domeinen of varianten van een eiwit.

Structuur-functie studies in muis embryonale stamcellen gebruiken recombinase-gemedieerde cassette uitwisseling

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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