Questo lavoro è stato sostenuto dal Ministerio de Educación y spagnolo Ciencia (BIO 2006-11792) e la Generalitat de Catalunya (2005-SGR00437). Gli autori ringraziano Marc Puigcerver e Jonatan Lucas per la loro assistenza tecnica per la preparazione dei mezzi. Tutto il personale da d&#39;Servei Estabulari è riconosciuto per la stabulazione degli animali, e soprattutto Juan Jose Martin e Javier Benito per il loro contributo supplementare a registrare parte di questo video. BCN è un collega del portoghese Fundação para a Ciência e Tecnologia e SG è un compagno dello spagnolo Ministerio de Educación y Ciencia.

La cura degli animali e le procedure sono state condotte secondo le linee guida e regolamenti approvati dal Comitato Etico per animali e la ricerca umana della Universitat Autònoma di Barcellona, ​​in Spagna. I. ovociti collezione <ol><li> Controllare e selezionare criceti femmina (Mesocricetus auratus; 8-12 settimana di età) del ceppo d&#39;oro siriano per la presenza di uno scarico di spessore vaginale (scarica fase postovulatoria). </li><li> Indurre femmine selezionati per superovulate mediante iniezione intraperitoneale di 40 UI di PMSG (Pregnant Mare Serum gonadotropina), seguita da 40 UI di hCG (gonadotropina corionica umana) 60 ore dopo. </li><li> Euthanize criceti femmina 16 ore dopo la somministrazione di hCG in una camera di anidride carbonica. </li><li> Isolare ovidotti dalle femmine, come mostrato nel video e trasferirli su una goccia di KSOM-H media. </li><li> Raccogliere ovocita-cumulo complessi strappando l&#39;ampolla ovidotto sotto un microscopio stereoscopico in una goccia di ialuronidasi (156 U / ml) a 37 ° C. Ovociti sarà liberato da cellule del cumulo in circa 5 minuti. </li><li> Pick up ovociti da ialuronidasi e lavarli due volte in KSOM-H media. </li></ol> Per la raccolta di embrioni di topo, topi di sesso femminile (Mus musculus, 6-8 settimane di età) sono indotti a superovulate mediante iniezione intraperitoneale di 5 UI di PMSG seguito da 5 UI di hCG dopo 48 ore e accoppiati con topi di sesso maschile. Le femmine sono sacrificati per dislocazione cervicale a 24-26, 44-46 o 54-56 ore dopo la somministrazione di hCG per ottenimento dei pronuclei, 2-4-cell o cellule di embrioni fase, rispettivamente. Raccolta di embrioni pronuclei viene eseguita esattamente come descritto per ovociti di criceto. Embrioni nelle fasi di 2-celle e 4-celle vengono raccolti svuotando la ovidotti con KSOM-H media [6]. Nota: A seguito di questo protocollo circa 20 o 30 ovociti / embrioni possono essere raccolti da ogni femmina. II. Preparazione di gelo e disgelo soluzioni <ol><li> Preparare la soluzione mi diluendo 0,57 ml di propilenglycol in 5 ml di KSOM-H (1,5 M di propilenglycol). </li><li> Preparare la soluzione disgelo con l&#39;aggiunta di 2 ml di KSOM-H ad un tubo con 0,2052 g di saccarosio (0,3 M di saccarosio). </li><li> Preparare la soluzione di congelamento con l&#39;aggiunta di 2 ml di soluzione che ho ad un tubo con 0,0684 g di saccarosio (1,5 M di saccarosio propilenglycol e 0,1 M). </li></ol> Nota: I Solution e il congelamento e scongelamento soluzioni devono essere preparate poco prima del loro utilizzo. Tuttavia, la determinata quantità di saccarosio da utilizzare per la preparazione delle soluzioni di congelamento e scongelamento può essere ponderata in anticipo e conservati in 3 ml tubi a temperatura ambiente per diversi mesi. III. Protocollo di congelamento <ol><li> Ovociti trasferimento / embrioni dal KSOM-H medio-una goccia di soluzione di I e incubare per 7-15 minuti a temperatura ambiente. </li><li> Nel frattempo, preparare un piatto di 90 millimetri di Petri per caricare il cannucce mettendo 90 gocce ml di congelamento e scongelamento soluzioni come viene mostrato nel video (1 goccia di ogni soluzione / paglia). </li><li> Trasferire il numero desiderato di ovociti / embrioni per le gocce soluzione di congelamento. Nota: Di solito trasferimento tra 5-30 ovociti o embrioni / drop. </li><li> Allegare una cannuccia in un sistema controllato aspirazione bocca e caricarlo seguendo questo ordine: soluzione che, d&#39;aria, con la caduta di congelamento degli ovociti, aria, calo di scongelamento, aria. </li><li> Una volta la paglia viene caricato, mantenere l&#39;aspirazione fino a quando la prima goccia introdotto (soluzione che ho) raggiunge il polimero alla fine superiore della paglia e tenute questo fine. Thermo-seal l&#39;altra estremità della paglia o di tenuta con un tappo in plastica. </li><li> Accendere il congelatore programmabile e selezionare il programma di congelamento. Il programma di congelamento utilizzato in questo protocollo si basa sulla velocità di raffreddamento originariamente pubblicato da Lassalle et al. [7]: <ul><li> 2 ° C / min dalla temperatura ambiente a -7 ° C 0 ° C / min per 10 minuti per consentire l&#39;equilibrio di temperatura e di induzione di semina (vedi sotto) </li><li> 0,3 ° C / min da -7 ° C a -30 ° C 0 ° C / min per 2 minuti per consentire l&#39;equilibrio di temperatura </li><li> 35 ° C / min da -30 ° C a -130 ° C </li></ul></li><li> Caricare le cannucce sugli slot del titolare del congelatore programmabile, assicurandosi che la parte della paglia con la goccia di soluzione di scongelamento è rivolto verso l&#39;esterno. Avviare il programma di congelamento. </li><li> Quando la temperatura del campione raggiunge i -7 ° C ed il raffreddamento è in attesa, effettuare la semina manuale: tuffo la pinza in azoto liquido, tirare i titolari un po &#39;fuori dal freezer per esporre la parte del cannucce contenente le gocce di soluzione di scongelamento, e subito tocco questa parte del cannucce con le pinze. Ripetere per ogni titolare e ogni paglia nel supporto, e poi lasciare che il programma di congelamento fino alla fine. </li><li> Quando il programma è completato, i titolari tuffo in azoto liquido. </li><li> Rimuovere le cannucce daltitolari e loro trasferimento in un alberello con etichetta. </li><li> Infine, memorizzare i alberello all&#39;interno di un serbatoio di azoto liquido. </li></ol> IV. Scongelamento protocollo <ol><li> Rimuovere la cannuccia dal serbatoio di azoto liquido. </li><li> Mantenere la paglia a temperatura ambiente per 40 secondi. </li><li> Mettere a bagno la paglia in un bagno d&#39;acqua a 30 ° C durante 40 secondi. </li><li> Togliere la spina dalla paglia o tagliare il termosaldata punta. </li><li> Svuotare la paglia in un 35-mm piastra di Petri e mescolare delicatamente le due gocce contenenti le soluzioni di congelamento e scongelamento. </li><li> Lascia ovociti / embrioni nella miscela per 15 minuti a temperatura ambiente. </li><li> Trasferimento ovociti / embrioni di un calo fresco di KSOM-H per altri 15 minuti a 37 ° C. </li><li> Ovociti / embrioni sono ora pronti per ulteriori manipolazioni. </li></ol> Nota: Durante i passi 6 e 7 si dovrebbe vedere che gli ovociti scongelati / embrioni lentamente si gonfiano a causa di reidratazione. V. Rappresentante Risultati L&#39;utilizzo del protocollo di crioconservazione qui riportati si traduce in un tasso di sopravvivenza di circa il 95% per ovociti di criceto e del&gt; 90%, 85% e 80% per il mouse B6CBAF1 embrioni al pronucleo, 2-cell stadi e 4-celle, rispettivamente. Quando sono embrioni di topo coltivati ​​in vitro in KSOM dopo lo scongelamento, almeno 70 al 80% dovrebbe arrivare allo stadio di blastocisti in condizioni di coltura ottimali. Si raccomanda di includere un gruppo di controllo (paglia) di ovociti / embrioni in ogni esperimento di congelamento e di scongelamento questo gruppo prima del resto degli ovociti / embrioni vengono utilizzati per ulteriori manipolazioni. Se i tassi di sopravvivenza per il gruppo di controllo sono inferiori alle attese, disgelo un&#39;altra paglia dello stesso lotto. Se i tassi di sopravvivenza per questo secondo gruppo sono bassi, scartare il restante cannucce da questo lotto di congelamento.

<ol>
	<li>Whittingham, D. G., Leibo, S. P., Mazur, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+of+mouse+embryos+frozen+to+-196+and+-269+&#176;C.">Survival of mouse embryos frozen to -196 and -269 &#176;C.</a> Science. 178, 411-414 (1972).</li><li>Wilmut, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cooling+rate,+warming+rate,+cryoprotective+agent+and+stage+of+development+on+survival+of+mouse+embryos+during+freezing+and+thawing.">The effect of cooling rate, warming rate, cryoprotective agent and stage of development on survival of mouse embryos during freezing and thawing.</a> Life Sci. 11, 1071-1079 (1972).</li><li>Parkening, T. A., Tsunoda, Y., Chang, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+various+low+temperatures,+cryoprotective+agents+and+cooling+rates+on+the+survival,+fertilizability+and+development+of+frozen-thawed+mouse+eggs.">Effects of various low temperatures, cryoprotective agents and cooling rates on the survival, fertilizability and development of frozen-thawed mouse eggs.</a> J. Exp. Zool. 197, 369-374 (1976).</li><li>Schneider, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryobiological+principles+of+embryo+freezing.">Cryobiological principles of embryo freezing.</a> J. In Vitro Fert. Embryo Transf. 3, 3-9 (1986).</li><li>Vajta, G., Kuwayama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+cryopreservation+systems.">Improving cryopreservation systems.</a> Theriogenology. 65, 236-244 (2006).</li><li>Duselis, A. R., Vrana, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrieval+of+mouse+oocytes.">Retrieval of mouse oocytes.</a> JoVE. 3, (2007).</li><li>Lassalle, B., Testart, J., Renard, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryo+features+that+influence+the+success+of+cryopreservation+with+the+use+of+1,2+propanediol.">Human embryo features that influence the success of cryopreservation with the use of 1,2 propanediol.</a> Fertil. Steril. 44, 645-651 (1985).</li><li>Ib&#225;&#241;ez, E., Grossmann, M., Vidal, F., Egozcue, J., Santal&#243;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+caprine+beta-lactoglobulin+transgenic+mouse+embryos.">Cryopreservation of caprine beta-lactoglobulin transgenic mouse embryos.</a> Cryobiology. 35, 290-298 (1997).</li><li>Dinnyes, A., Wallace, G. A., Rall, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+genotype+on+the+efficiency+of+mouse+embryo+cryopreservation+by+vitrification+or+slow+freezing.">Effect of genotype on the efficiency of mouse embryo cryopreservation by vitrification or slow freezing.</a> Mol. Reprod. Dev. 40, 429-435 (1995).</li><li>Tateno, H., Kamiguchi, Y., Mikamo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+freezing+and+thawing+method+of+hamster+oocytes+designed+for+both+the+penetration+test+and+chromosome+assay+of+human+spermatozoa.">A freezing and thawing method of hamster oocytes designed for both the penetration test and chromosome assay of human spermatozoa.</a> Mol. Reprod. Dev. 33, 202-209 (1992).</li><li>Biggers, J., McGuinnis, L. K., Raffin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+acids+and+preimplantation+development+of+the+mouse+in+the+protein-free+KSOM.">Amino acids and preimplantation development of the mouse in the protein-free KSOM.</a> Biol. Reprod. 63, 281-293 (2000).</li></ol>

Con questo protocollo e ovociti di criceto embrioni di topo possono essere crioconservati con successo. Una volta congelati, gli ovociti / embrioni possono essere conservati in serbatoi di azoto liquido a tempo indeterminato e recuperato in qualsiasi momento o luogo desiderato. Questo offre il vantaggio di avere campioni quasi pronto per l&#39;uso in caso di necessità. D&#39;altra parte, questo protocollo può essere utilizzato anche per generare criobanche embrione di ceppi di topi di valore (come linee transgeniche), come alternativa eco...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>French type mini-straw 0.25 ml</td>
<td>&nbsp;</td>
<td>Mini-tub</td>
<td>13407/0010</td>
<td>&nbsp;</td>
<tr>
<td>hCG</td>
<td>&nbsp;</td>
<td>Farma-Lepori, Spain</td>
<td>749036.4</td>
<td>&nbsp;</td>
<tr>
<td>Hyaluronidase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H-3884</td>
<td>&nbsp;</td>
<tr>
<td>KSOM-H medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Ref. [11]</td>
</tr>
<tr>
<td>Liquid Nitrogen </td>
<td>&nbsp;</td>
<td>Air Liquide</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plastic plugs</td>
<td>&nbsp;</td>
<td>Mini-tub</td>
<td>19046</td>
<td>&nbsp;</td>
<tr>
<td>PMSG</td>
<td>&nbsp;</td>
<td>Intervet, Spain</td>
<td>1.614/8447</td>
<td>&nbsp;</td>
<tr>
<td>Propilenglycol</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>82280</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>107687</td>
<td>&nbsp;</td>
<tr>
<td>Surgical Scissors</td>
<td>&nbsp;</td>
<td>Aesculap</td>
<td>BC-060R; BG-061R</td>
<td>&nbsp;</td>
<tr>
<td>Thermo-sealer</td>
<td>&nbsp;</td>
<td>SIZ</td>
<td>220</td>
<td>&nbsp;</td>
<tr>
<td>35 and 90 mm culture dishes</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>153066; 150350</td>
<td>&nbsp;</td>
<tr>
<td>10 ml tubes</td>
<td>&nbsp;</td>
<td>Greiner Bio-one</td>
<td>163160</td>
<td>&nbsp;</td>
<tr>
<td>Nitrogen tank</td>
<td>&nbsp;</td>
<td>MVE, Cryogenics</td>
<td>XC 43/28</td>
<td>&nbsp;</td>
<tr>
<td>Programmable Freezer</td>
<td>&nbsp;</td>
<td>Planner Products Ltd.</td>
<td>Kryo-10 Series III</td>
<td>&nbsp;</td>
<tr>
<td>Forceps</td>
<td>&nbsp;</td>
<td>Aesculap</td>
<td>BD-331R; BD-053R; OC-020R</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

collection and cryopreservation of hamster oocytes and mouse embryos

Embrioni e ovociti crioconservati con successo sono stati più di 30 anni fa, quando Whittingham<i&gt; Et al.</i&gt;<sup&gt; 1</sup&gt; E Wilmut<sup&gt; 2</sup&gt; Separatamente descritto embrioni di topo che potrebbero essere congelati e conservati a -196 ° C e, pochi anni dopo, Parkening<i&gt; Et al</i&gt;.<sup&gt; 3</sup&gt; Segnalato la nascita di piccoli vivi da fecondazione in vitro (IVF) di ovociti crioconservati. Da allora, l&#39;uso di tecniche di crioconservazione è rapidamente diffusa a diventare una componente essenziale nella pratica della umana e animale riproduzione assistita e nella conservazione delle risorse genetiche animali. Attualmente, ci sono due metodi principali utilizzati per crioconservare gli ovociti e gli embrioni: il congelamento lento e vitrificazione. Una vasta gamma di approcci sono stati utilizzati per cercare di migliorare sia le tecniche e milioni di animali e migliaia di bambini sono nati da embrioni crioconservati. Tuttavia, le carenze importanti associati alla crioconservazione devono ancora essere superate, in quanto formazione di cristalli di ghiaccio, effetti soluzione e shock osmotico sembrano causare cryoinjuries diversi post-scongelamento degli ovociti e degli embrioni. Congelamento lento con congelatori programmabili ha il vantaggio di usare basse concentrazioni di crioprotettori, che sono di solito associati a tossicità chimica e shock osmotico, ma la loro capacità di evitare la formazione di cristalli di ghiaccio a basse concentrazioni è limitata. Congelamento lento induce anche gli effetti surraffreddamento che deve essere evitato di usare la semina manuale o automatico<sup&gt; 4</sup&gt;. Nel processo di vetrificazione, alte concentrazioni di crioprotettori inibire la formazione di cristalli di ghiaccio e portare alla formazione di uno stato simile a vetro ceramico in cui è solidificato acqua, ma non ampliato. Tuttavia, a causa della tossicità del cyroprotectants alle concentrazioni utilizzate, gli ovociti / embrioni non può che essere esposti alla soluzione crioprotettore per un periodo di tempo molto breve e in una soluzione di volume minimo, prima sommergendo i campioni direttamente in azoto liquido<sup&gt; 5</sup&gt;. Negli ultimi dieci anni, vetrificazione è diventato più popolare perché è un metodo molto veloce in cui è richiesta nessuna attrezzatura costosa (congelatore programmabile). Tuttavia, il congelamento lento continua ad essere il metodo più utilizzato per ovocita / embrione crioconservazione. In questo video-articolo mostriamo, passo per passo, come raccogliere e lentamente congelare ovociti di criceto con elevate post-disgelo tassi di sopravvivenza. La stessa procedura può anche essere applicato con successo per congelare e scongelare embrioni di topo a diversi stadi di sviluppo preimpianto.

Watch this Scientific Journal Video about Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos at JoVE.com

Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

In questo video-articolo vi presentiamo un passo-passo dimostrazione su come raccogliere e crioconservare gli ovociti di criceto con elevate post-disgelo tassi di sopravvivenza. La stessa procedura può anche essere applicato con successo per congelare e scongelare embrioni di topo a diversi stadi di sviluppo preimpianto.

Raccolta e Crioconservazione degli ovociti e di embrioni di topo criceto

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse ...

collection-and-cryopreservation-of-hamster-oocytes-and-mouse-embryos

Research

JoVE Journal

Biology

16.8K Views.  Universitat Autonoma de Barcelona. In questo video-articolo vi presentiamo un passo-passo dimostrazione su come raccogliere e crioconservare gli ovociti di criceto con elevate post-disgelo tassi di sopravvivenza. La stessa procedura può anche essere applicato con successo per congelare e scongelare embrioni di topo a diversi stadi di sviluppo preimpianto.

Video: Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

Nuclear Transfer into Mouse Oocytes

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying development, differentiation and aging are epigenetic rather than genetic processes. The reversibility of these processes opens exciting perspectives in basic research, and in the more distant future, in regenerative medicine. In the mouse, embryonic stem cells can be derived from cloned preimplantation stage embryos. Such embryonic stem cells have the ability to give rise to all cell types of the adult organism. Importantly, these cells are genetically identical to the donor. If applicable to human, this would allow the derivation of stem cells from a patient. These cells could then be differentiated into the affected cell type of the patient and studied in vitro, or used to replace the damaged or missing cells. The study of nuclear transfer in the mouse remains important as it can inform us about the principles of nuclear reprogramming. This movie and the accompanying protocol are intended to help learning nuclear transfer in the mouse, a method initially developed in the group of Prof. Yanagimachi (WAKAYAMA et al. 1998).

This movie and the protocol are intended to help learning nuclear transfer.

Trasferimento nucleare in oociti mouse

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying ...

Ricerca

Biologia

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

Dissezione di 6,5 embrioni di topo dpc

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

Retrieval of Mouse Oocytes

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus Genetic Stock Center (http://stkctr.biol.sc.edu), we are characterizing the salient differences needed to develop this system.  A primary goal has been to optimize oocyte/early embryo retrieval.

This protocol illustrates the technique for extracting oocytes or early-stage fertilized embryos from the oviduct of mice. The ability to identify the infindibulum and insert a blunt end needle into it is essential to correctly performing the procedure.

Recupero degli ovociti mouse

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus ...

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video. 

In Utero iniezione intraventricolare e Elettroporazione di E15 embrioni di topo

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

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

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

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

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Imaging in vivo e trattamenti terapeutici in un modello di mouse ortotopico di cancro ovarico

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

Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats

Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage. Preimplantation embryos consist predominantly of water, and the avoidance of intracellular ice crystal formation during the cryopreservation process is of paramount importance to maintain embryo viability. Embryos are placed into a hypertonic solution (1.4 &#x2013; 1.5 M) of a cryoprotective agent (CPA) such as ethylene glycol (EG) or glycerol (GLYC) to create an osmotic gradient that facilitates cellular dehydration. After embryos reach osmotic equilibrium in the CPA solution, they are individually loaded in the hypertonic CPA solution into 0.25 ml plastic straws for freezing. Embryos are placed into a controlled rate freezer at a temperature of -6&deg;C. Ice crystal formation is induced in the CPA solution surrounding the embryo, and crystallization causes an increase in the concentration of CPA outside of the embryo, causing further cellular dehydration. Embryos are cooled at a rate of 0.5&deg;C/min, enabling further dehydration, to a temperature of -34&deg;C before being plunged into liquid nitrogen (-196&deg;C). Cryopreserved embryos must be thawed prior to transfer to a recipient (surrogate) female. Straws containing the embryos are removed from the liquid nitrogen dewar, held in room temperature air for 3 to 5 sec, and placed into a 37&deg;C water bath for 25 to 30 sec. Embryos cryopreserved in GLYC are placed into a 1 M solution of sucrose for 10 min for removal of the CPA before transfer to a recipient (surrogate) female. Embryos cryopreserved in EG, however, may be directly transferred to the uterus of a recipient.

Preimplantation embryos may be cryopreserved after placement into a hypertonic cryoprotective solution to cause cellular dehydration. After equilibration, ice crystal formation is induced in the solution surrounding the embryo. Further dehydration occurs as the embryo is slowly cooled to subzero temperatures before plunging into liquid nitrogen for storage.

Crioconservazione di embrioni preimpianto di bovini, ovini, e caprini

Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage.  Preimplantation embryos consist predominantly ...

Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic modifications and the number is consistently increasing year by year. Its technical development started with slow freezing methods in the 1970s1, then followed by vitrification methods developed in the late 1980s2. Generally, the latter technique is advantageous in its quickness, simplicity, and high survivability of recovered embryos. However, the cryoprotectants contained are highly toxic and may affect subsequent embryo development. Therefore, the technique was not applicable to certain strains of mice, even when the solutions are cooled to 4&deg;C to mitigate the toxic effect during embryo handling. At the RIKEN BioResource Center, more than 5000 mouse strains with different genetic backgrounds and phenotypes are maintained3, and therefore we have optimized a vitrification technique with which we can cryopreserve embryos from many different strains of mice, with the benefits of high embryo survival after vitrifying and thawing (or liquefying, more precisely) at the ambient temperature4.
Here, we present a vitrification method for mouse embryos that has been successfully used at our center. The cryopreservation solution contains ethylene glycol instead of DMSO to minimize the toxicity to embryos5. It also contains Ficoll and sucrose for prevention of devitrification and osmotic adjustment, respectively. Embryos can be handled at room temperature and transferred into liquid nitrogen within 5 min. Because the original method was optimized for plastic straws as containers, we have slightly modified the protocol for cryotubes, which are more easily accessible in laboratories and more resistant to physical damages. We also describe the procedure of thawing vitrified embryos in detail because it is a critical step for efficient recovery of live mice. These methodologies would be helpful to researchers and technicians who need preservation of mouse strains for later use in a safe and cost-effective manner.

An ethylene glycol-based vitrification method for mouse embryos is described. It is advantageous to other methods in its simplicity and low embryonic toxicity, and therefore can be broadly applicable to many strains of mice, including inbred and gene-modified mice.

Crioconservazione di embrioni di topo da glicole etilenico-Based Vetrificazione

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Mouse Sperm Cryopreservation and Recovery using the I&amp;middot;Cryo Kit

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

Crioconservazione del mouse Sperm and Recovery utilizzando il Kit · I Cryo

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

Meiotic mandrino valutazione in mouse Ovociti mettendo a tacere siRNA-mediata

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.

Here, we describe a method for warming vitrified human blastocysts, culturing them through the implantation period in vitro, digesting them into single cells and collecting early trophoblast cells for further investigation.

Raccolta a cella singola di cellule di trofoblasto in peri-impianto Stadio Embrioni Umani

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a ...