Die Forschung hier vorgestellte wurde von Charles River unterstützt. Die Autoren sind sehr dankbar, dass der gentechnisch veränderte Models and Services-Gruppe und der Embryologie Mitarbeiter für die Tierpflege und Hormon-Einspritzung.

1. Maus Spermien Kryokonservierung <ol><li> Für jede Zeile kryokonserviert werden, bereiten Sie die folgenden Punkte, bevor Anfang. <ol><li> 1 gut einer 4-Well Platte mit 240 l Kit CPA </li><li> 15 Samen Kryokonservierung Strohhalme wie folgt <ol><li> Schließen Sie das 0,25 ml Pailletten, die 1 ml Spritzen mit dem Wattebausch am nächsten an der Spritze. In jedem Strohhalm, Last 8 cm (ca. 160 ul) IVF-Medium, dann 1 cm Luft. Beschriften Sie die Strohhalme mit den Namen der Linie erhalten bleiben. </li></ol></li></ol></li><li> LN 2 sollte 15 cm tief in den Schaumstoff-Box werden. Schließen Sie den Deckel. </li><li> Euthanize 2 männliche Mäuse aus jeder Zeile zu kryokonserviert werden. Mäuse sollten zwischen 10 und 60 Wochen alt sein. Entfernen Sie die beiden kaudalen Nebenhoden zusammen mit der Samenleiter von jeder Maus. </li><li> Setzen Sie den Nebenhoden in die zuvor vorbereiteten CPA-haltigen Vertiefung einer 4-Well Schale (Schritt 1.1). </li><li> Machen Sie 5-7 Längsschnitte in jedem Nebenhoden und sanftSqueeze jedem Samenleiter Spermien verdrängen. </li><li> Schütteln Sie die CPA und Nebenhoden Mischung von Hand vorsichtig bei Raumtemperatur für 3-5 Minuten. Damit können Spermien schwimmen. </li><li> Mit dem zuvor vorbereiteten Stroh, absaugen 5 mm (ca. 10 ul) der Spermien Federung und dann 1 cm Luft. </li><li> Wiederholen Sie diesen Vorgang mit bis zu insgesamt 15 Strohhalme. Laden und Dichtung Strohhalme innerhalb von 10 Minuten für die besten Spermien überleben. </li><li> Heißsiegel beiden Seiten des Stroh sorgfältig. Legen Sie die Strohhalme in den Gefrierpunkt Kanister mit dem Sperma Säule zuerst. </li><li> Bringen Sie die 1 ml Pipette als Teil des Kits zu den Kanister mit dem Griff erstrecken. Setzen Sie den Kanister in LN 2 in der Schaum-Box durch das Loch in den Deckel. Lassen Sie den Kanister vertikal Schwimmer in der LN 2 für 10 Minuten. </li><li> Tauchen Sie den Kanister in das LN2. Das Sperma wird nun kryokonserviert und können zu langfristigen LN 2-Speicherung verschoben werden. </li><li> Der Behälter sollte nicht verwendet werden fürdie nächste Zeile, bis es sich auf Raumtemperatur erwärmt. </li></ol> 2. Maus Superovulation <ol><li> Im Allgemeinen benutzen jungen weiblichen Mäusen des gleichen Hintergrund wie der Samenspender. </li><li> Mäuse sollten ~ 12 g (3-4 Wochen alt) werden. </li><li> Führen Sie eine intraperitoneale Injektion mit 5-10 IE Pregnant mare serum-Gonadotropin (PMSG). </li><li> Warten Sie 46-48 Stunden. </li><li> Führen Sie eine intraperitoneale Injektion mit 5-10 IU humanes Chorion-Gonadotropin (hCG). </li><li> Ernte-Oozyten 14-16 h nach hCG-Injektion, wie unten beschrieben. </li></ol> 3. Mausspermien Erholung und IVF <ol><li> Vor IVF, für jeden Strohhalm aufgetaut werden, bereiten die folgenden und für mindestens eine Stunde ins Gleichgewicht in einem 37 ° C CO 2-Inkubator. <ol><li> Sperm Inkubation Schüssel: eine 35 mm Petrischale mit einem 90 ul Tropfen Sperma Behandlung Medium (STM) mit Öl bedeckt. </li><li> Cutting Gericht: Für Eizellenentnahme von maximal 10 superovulated Weibchen, einer 35 mm Petrischale mit 3 ml IVF Medium. </li><li> IVF gut: Für jede Gruppe von 5 superovulierten Weibchen für IVF, eine Vertiefung einer 4 gut Schüssel mit 500 ul IVF Medium (wenn Sie mit 10 superovulierten Weibchen sind, zum Beispiel, Sie erhalten 2 Brunnen benötigen...) </li><li> Washing Schüssel: eine 35 mm Petrischale mit 3 ml Kalium-simplex optimierten Medium (KSOM) zum Waschen jeder Vertiefung der 4-und IVF Gericht verwendet </li><li> Kulturschale: eine 35 mm Petrischale mit 50 ul Tropfen KSOM Medium mit Öl für die Embryo-Kultur aus jedem Well der IVF Schüssel abgedeckt. </li></ol></li><li> Nehmen Sie einen Strohhalm aus LN 2 und in einem 37 ° C Wasserbad für 2-3 Minuten. </li><li> Entfernen Sie das Stroh aus dem Wasserbad und wischen, um überschüssiges Wasser zu entfernen. Machen Sie den ersten Schnitt in der Nähe der ersten Luftsäule nächst der Wattebausch. Schneiden Sie das Stroh in der IVF-Medium gibt es noch eine Luftsäule sichtbar, bevor die Spermien Spalte werden. </li><li> Dann schneiden Sie das Ende des Strohs in den sZWEITEN Luftsäule, wobei darauf zu vermeiden, schneiden die Spermien Spalte. Cut bei einem 45 ° Winkel - das macht es viel einfacher, das Sperma absaugen. </li><li> Saugen Sie die Spermien Suspension aus dem distalen Ende des Strohs mit einer 200 ul Pipette. </li><li> Legen Sie die Spermien Suspension in der Mitte des STM Drop und inkubieren Sie die Schale für 45-60 Minuten in einem 37 ° C 5% CO 2-Inkubator. </li><li> Während die Spermien Inkubation sezieren die Eileiter der superovulierten Weibchen in die Schneiden Gericht. Verwenden Sie eine 30 g Nadel oder Pinzette zu jedem Eileiter reißen, die Freigabe der Kumulus-Eizell Komplexe (KOK). Vermeiden Sie Fett und Blut in der IVF-Medium wie verschmutzte Medien für zusätzliche Waschschritte macht. </li><li> Nachdem alle die Eileiter wurden abgerissen, übertragen Sie die KOK von 5 Hündinnen zu jedem IVF Brunnen. Die Zahl der KOK durch Superovulation produziert ist sehr belasten abhängig. </li><li> Sammeln 10-15 ul von Spermien aus der Peripherie des STM Drop und befruchten IVF und etwa 14-16 Stunden post hCG injection. Wiederholen Sie die Befruchtung für jeden gut genutzt. Finale Spermienkonzentration werden ca. 1 Mio. / ml sein. </li><li> Cokultur Spermien und Eizellen für 4-6 Stunden in einem 37 ° C 5% CO 2-Inkubator. </li><li> Waschen Sie die Embryonen mit KSOM mindestens 2 Mal und Kultur in KSOM Tropfen für die Entwicklung in einem 37 ° C 5% CO 2-Inkubator. </li><li> IVF-Rate wurde in zwei Zell-Embryonen aus insgesamt Eizellen nach Übernacht-Kultur verwendet berechnet. </li></ol> 4. Repräsentative Ergebnisse Sperma von 5 Charles River Maus Inzuchtstämme wurde eingefroren. Schutz Verhältnisse sind, indem gefrorenen Sperma Beurteilung Werte von frischem Sperma Bewertung berechnet. Schutz-Verhältnisse von Spermien-Motilität wurden 60,2%, 81,3%, 78,6%, 66,5%, 61,0% für C57BL/6NCrl, 129S2/SvPasCrl, FVB / NCrl, DBA/2NCrl und BALB / cAnNCrl jeweils. Schutz-Verhältnisse für schnellen progressiven Motilität wurden 47,3%, 69,4%, 57,0%, 52,8%, 54,1% in C57BL/6NCrl, 129S2/SvPas<html> CRL, FVB / NCrl, DBA/2NCrl und BALB / cAnNCrl bzw. (Abb. 1). Die IVF Raten mit aufgetauten Spermien wurden 55,7%, 26,4%, 87,3%, 87,8% und 26,2% in C57BL/6NCrl, 129S2/SvPasCrl, FVB / NCrl, DBA/2NCrl und BALB / cAnNCrl bzw. (Abb. 2) . Über einen 1,5 Jahre Zeitraum wurden 49 GM Mauslinien durch Spermien Kryokonservierung archiviert. Diese Linien wurden später erfolgreich wiederhergestellt (wie Live-Welpen geboren definiert) durch IVF in 4 weiblichen Stämmen (C57BL / 6, BALB / c, DBA / 1 und 129Sv) (Abb. 3). <img alt="Abbildung 1." src="/files/ftp_upload/3713/3713fig1.jpg" /> Abbildung 1. Der Schutz Ratios von Spermien-Motilität und schnellen progressiven Motilität in Mäuse <img alt="Abbildung 2." src="/files/ftp_upload/3713/3713fig2.jpg" /> Abbildung 2. In-Vitro-Fertilisation mit aufgetauten Sperma in Mäuse 3713fig3.jpg &quot;/&gt; Abbildung 3. In-vitro-Fertilisation mit aufgetauten Sperma in GM Mäuse

<ol>
	<li>Tada, N., Sato, M., Yamanoi, J., Mizorogi, T., Kasai, K., Ogawa, S. <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+mouse+spermatozoa+in+the+presence+of+raffinose+and+glycerol.">Cryopreservation of mouse spermatozoa in the presence of raffinose and glycerol.</a> J. Reprod. Fertil. 89, 511-516 (1990).</li><li>Yokoyama, M., Akiba, H., Katsuki, M., Nomura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+normal+young+following+transfer+of+mouse+embryos+obtained+by+in+vitro+fertilization+using+cryopreserved+spermatozoa.">Production of normal young following transfer of mouse embryos obtained by in vitro fertilization using cryopreserved spermatozoa.</a> Jikken Dobutsu. 39, 125-128 (1990).</li><li>Sztein, J. M., Farley, J. S., Young, A. F., Mobraaten, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motility+of+cryopreserved+mouse+spermatozoa+affected+by+temperature+of+collection+and+rate+of+thawing.">Motility of cryopreserved mouse spermatozoa affected by temperature of collection and rate of thawing.</a> Cryobiology. 35, 46-52 (1997).</li><li>Thornton, C. E., Brown, S. D., Glenister, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+numbers+of+mice+established+by+in+vitro+fertilization+with+cryopreserved+spermatozoa:+implications+and+applications+for+genetic+resource+banks,+mutagenesis+screens,+and+mouse+backcrosses.">Large numbers of mice established by in vitro fertilization with cryopreserved spermatozoa: implications and applications for genetic resource banks, mutagenesis screens, and mouse backcrosses.</a> Mamm. Genome. 10, 987-992 (1999).</li><li>Sztein, J. M., Farley, J. S., Mobraaten, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+fertilization+with+cryopreserved+inbred+mouse+sperm.">In vitro fertilization with cryopreserved inbred mouse sperm.</a> Biol. Reprod. 63, 1774-1780 (2000).</li><li>Liu, L., Nutter, L. M., Law, N., McKerlie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+freezing+and+in+vitro+fertilization+in+three+substrains+of+C57BL/6+mice.">Sperm freezing and in vitro fertilization in three substrains of C57BL/6 mice.</a> J. Am. Assoc. Lab. Anim. Sci. 48, 39-43 (2009).</li><li>Nakagata, N. <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+mouse+spermatozoa+and+in+vitro+fertilization.">Cryopreservation of mouse spermatozoa and in vitro fertilization.</a> Methods. Mol. Biol. 693, 57-73 (2011).</li><li>Mazur, P., Koshimoto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+intracellular+ice+formation+the+cause+of+death+of+mouse+sperm+frozen+at+high+cooling+rates.">Is intracellular ice formation the cause of death of mouse sperm frozen at high cooling rates.</a> Biol. Reprod. 66, 1485-1490 (2002).</li><li>Jin, B., Yamasaki, C., Yamada, N., Seki, S., Valdez, D. M., Kasai, M., Edashige, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+by+which+mouse+spermatozoa+are+injured+during+freezing.">The mechanism by which mouse spermatozoa are injured during freezing.</a> J. Reprod. Dev. 54, 265-269 (2008).</li><li>Visconti, P. E., Westbrook, V. A., Chertihin, O., Demarco, I., Sleight, S., Diekman, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+signaling+pathways+involved+in+sperm+acquisition+of+fertilizing+capacity.">Novel signaling pathways involved in sperm acquisition of fertilizing capacity.</a> J. Reprod. Immunol. 53, 133-150 (2002).</li><li>Ostermeier, G. C., Wiles, M. V., Farley, J. S., Taft, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserving,+distributing+and+managing+genetically+modified+mouse+lines+by+sperm+cryopreservation.">Conserving, distributing and managing genetically modified mouse lines by sperm cryopreservation.</a> PLoS ONE. 3, e2792-e2792 (2008).</li></ol>

Die Autoren sind Mitarbeiter von Charles River, der Hersteller des Kits.

Seit 1990 ist die grundlegende Sperma Einfrieren Protokoll mit 18% Raffinose und 3% Magermilch erwiesen in viele Stämme von Mäusen zuverlässig und eine große Anzahl von Mauslinien wurden 1,2,3,4,5,6,7 kryokonserviert. Die wichtigsten Ursachen für Schäden an Spermien während der Frost-Tau-Prozess haben mit Eisbildung 8,9 und reaktiven Sauerstoff-Spezies 10 in Verbindung gebracht. Supplementierung mit Radikalfängern, wie Aminosäuren und Reduktionsmittel, wie z. B. Monothioglycerol, in dem Einfriermedium wurde untersucht und bewiesen deutlich zu erhöhen IVF-Rate mit aufgetauten Samenzellen in Inzuchtstämme einschließlich der C57BL / 6 11,6. Im vorliegenden Protokoll, entwickelten wir einen neuen I • Cryo-Kit für Mausspermien Kryokonservierung durch die Optimierung der grundlegenden CPA mit handelsüblichen Antioxidantien und Eis-Blocker und durch Veränderung der Spermien Einfrieren und IVF-Verfahren. Schutz-Verhältnisse von Spermien-Motilität(&gt; 60%) und schnellen progressiven Motilität (&gt; 45%) wurden für die Spermien von fünf Inzucht-Mausstämmen mit dem I • Cryo-Kit eingefroren gesehen. Die 2-Zell-Stadium der Embryonalentwicklung Rate mit aufgetauten Samenzellen in 5 Maus Inzuchtstämme war deutlich im Vergleich zu dem mit den grundlegenden CPA 11,6 verbessert. In Verwendung des Kits erstellt der Benutzer eine konzentrierte Sperma Suspension durch das Einfrieren von Spermien 2 Rüden für jede Zeile. Mit nur Einfrieren 15 Strohhalme der Prozess ist schnell, einfach und belegt weniger Speicherplatz für kurze oder langfristige Lagerung. Die meiste Zeit kann Mauslinien durch Auftauen nur 1 oder 2 Strohhalme wiederhergestellt werden.

<table border="1">
	<tbody>
		<tr>
			<th>
				Materials</th>
			<th>
				Supplier</th>
			<th>
				Catalogue number</th>
			<th>
				Comments</th>
		</tr>
		<tr>
			<td>CPA</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>STM</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>IVF medium</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>0.25 ml plastic straw</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>Sperm freezing canister</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>Cane and goblet</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma</td>
			<td>M5310</td>
			<td>Should be embryo tested</td>
		</tr>
		<tr>
			<td>KSOM medium</td>
			<td>Millipore</td>
			<td>MR-106-D</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>35 mm Petri dish</td>
			<td>Falcon</td>
			<td>351008</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heat sealer</td>
			<td>ABTEC</td>
			<td>TISH-200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>4-well multidish</td>
			<td>Nunc</td>
			<td>73521-424</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pipettor</td>
			<td>Gilson</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>Various</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>37&deg;C + 5% CO2 incubator</td>
			<td>Various</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LN2 storage system</td>
			<td>Various</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>37&deg;C water bath</td>
			<td>VWR</td>
			<td>89032-196</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>SMZ800</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>hCG</td>
			<td>Intervet</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PMSG</td>
			<td>EMDChemicals</td>
			<td>367222</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1cc syringe w/ 26G 3/8 needle</td>
			<td>BD</td>
			<td>BD309625</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micro dissecting scissors</td>
			<td>Roboz</td>
			<td>RS-5602</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>30 gauge &frac12; needle</td>
			<td>BD</td>
			<td>305106</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Roboz</td>
			<td>RS-6802</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Roboz</td>
			<td>RS-5135, RS-5110, RS-5005</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

mouse sperm cryopreservation and recovery using the i&middot;cryo kit

Tausende von neuen gentechnisch veränderten (GV) Stämme von Mäusen haben seit dem Aufkommen der Transgenese und Knockout-Technologien geschaffen worden. Viele dieser wertvollen Tiere existieren nur als lebende Tiere, ohne Backup-Plan für den Notfall. Die Kryokonservierung von Embryonen können dieses Backup bieten, ist aber teuer, kann ein langwieriges Verfahren sein, und erfordert in der Regel eine große Anzahl von Tieren für den Erfolg. Seit der Entdeckung, dass Mausspermien erfolgreich mit einer grundlegenden kryoprotektive Agent (CPA), bestehend aus 18% Raffinose und 3% Magermilch kryokonserviert werden, hat Sperma Kryokonservierung zu einem akzeptablen und kostengünstiges Verfahren für die Archivierung, Verteilung und Rückgewinnung dieser wertvollen Stämme . Hier zeigen wir ein neu entwickeltes I • Cryo-Kit für Mausspermien Kryokonservierung. Sperma aus fünf am häufigsten verwendeten Stämme von Inzucht-Mäusen wurden eingefroren mit diesem Kit und dann wiederhergestellt. Besserer Schutz-Verhältnisse von Spermien-Motilität (&gt;60%) und schnellen progressiven Motilität (&gt; 45%) im Vergleich zur Kontrollgruppe (unverwässert CPA) wurden für die Spermien mit diesem Kit in 5 Inzucht-Mausstämmen eingefroren gesehen. Zwei-Zell-Stadium der Embryonalentwicklung nach IVF mit dem Sperma gewonnen wurde konsequent in allen 5 untersuchten Mausstämme verbessert. Über einen 1,5 Jahre Zeitraum wurden 49 GM Mauslinien durch Spermien Kryokonservierung mit dem I • Cryo-Kit archiviert und später durch IVF gewonnen.

Watch this Scientific Journal Video about Mouse Sperm Cryopreservation and Recovery using the IÃ‚Â·Cryo Kit at JoVE.com

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

Hier zeigen wir den neu entwickelten I • Cryo-Kit für Mausspermien Kryokonservierung. Zwei-Zell-Stadium der Embryonalentwicklung mit aufgetauten Sperma wurde konsequent in 5 Mausstämme mit dem Einsatz dieses Kits verbessert. Über einen 1,5 Jahre Zeitraum wurden 49 genetisch veränderte Mauslinien durch Spermien Kryokonservierung mit dem I • Cryo-Kit archiviert und später erfolgreich durch IVF gewonnen.

Maus Sperma Kryokonservierung und Recovery mit der I · 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 ...

mouse-sperm-cryopreservation-and-recovery-using-the-icryo-kit

Research

JoVE Journal

Biology

39.5K Aufrufe.  Charles River. Hier zeigen wir den neu entwickelten I • Cryo-Kit für Mausspermien Kryokonservierung. Zwei-Zell-Stadium der Embryonalentwicklung mit aufgetauten Sperma wurde konsequent in 5 Mausstämme mit dem Einsatz dieses Kits verbessert. Über einen 1,5 Jahre Zeitraum wurden 49 genetisch veränderte Mauslinien durch Spermien Kryokonservierung mit dem I • Cryo-Kit archiviert und später erfolgreich durch IVF gewonnen.

Serie: Mouse Sperm Cryopreservation and Recovery using the I·Cryo Kit

Harvesting Sperm and Artificial Insemination of Mice

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals. Peromyscus species are used in a wide range of research including toxicology, epidemiology, ecology, behavioral, and genetic studies. Here they provide a useful model for demonstrations of artificial insemination. 

Methods similar to those displayed here have previously been used in several deer mouse studies, yet no detailed protocol has been published. Here we demonstrate the basic method of artificial insemination. This method entails extracting the testes from the rodent, then isolating the sperm from the epididymis and vas deferens. The mature sperm, now in a milk mixture, are placed in the female&#x2019;s reproductive tract at the time of ovulation. Fertilization is counted as day 0 for timing of embryo development. Embryos can then be retrieved at the desired time-point and manipulated.

Artificial insemination can be used in a variety of rodent species where exact embryo timing is crucial or hard to obtain. This technique is vital for species or strains (including most Peromyscus) which may not mate immediately and/or where mating is hard to assess. In addition, artificial insemination provides exact timing for embryo development either in mapping developmental progress and/or transgenic work. Reduced numbers of animals can be used since fertilization is guaranteed. This method has been vital to furthering the Peromyscus system, and will hopefully benefit others as well.

This protocol demonstrates methods for extracting sperm from the testes of males and then inseminating female mice. This procedure is useful when precise time is needed in developmental studies as well as transgenic work.

Harvesting Sperma und künstliche Besamung von Mice

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals.  Peromyscus species are used in a wide range of research ...

Forschung

Biologie

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.

Purifying Plasmid-DNA aus Bakterien-Kolonien mit dem Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial ...

Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.

Immunhistochemie: Paraffin Schnitte mit der Vectastain ABC Kit von Vector Labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific ...

RNA Extraction from Neuroprecursor Cells Using the Bio-Rad Total RNA Kit

RNA Extraktion aus Neuroprecursor Zellen unter Verwendung des Bio-Rad Total RNA Kit

Rapid Genotyping of Mouse Tissue Using Sigma's Extract-N-Amp Tissue PCR Kit

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is determined. Conventional preparation of tissues for PCR-ready DNA often take several hours to days, depending on the tissue sample. The genotype of the sample may thus be delayed for several days, which is not an option for many different types of experiments. Here we demonstrate the complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR Kit. First, we demonstrate the fifteen-minute extraction of DNA from the tissue sample. Then, we demonstrate the real time read-out of the PCR amplification of the sample, which allows for the identification of a positive sample as it is being amplified. Together, the rapid extraction and real-time readout allow for a prompt identification of genotype of a variety different types of tissues through the reliable method of PCR.

The complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, is done in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR kit.

Schnelle Genotypisierung von Maus Tissue Mit Sigma Extract-N-Amp Tissue PCR Kit

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is ...

Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse embryos could be frozen and stored at -196 &deg;C and, a few years later, Parkening et al. 3 reported the birth of live offspring resulting from in vitro fertilization (IVF) of cryopreserved oocytes. Since then, the use of cryopreservation techniques has rapidly spread to become an essential component in the practice of human and animal assisted reproduction and in the conservation of animal genetic resources. Currently, there are two main methods used to cryopreserve oocytes and embryos: slow freezing and vitrification. A wide variety of approaches have been used to try to improve both techniques and millions of animals and thousands of children have been born from cryopreserved embryos. However, important shortcomings associated to cryopreservation still have to be overcome, since ice-crystal formation, solution effects and osmotic shock seem to cause several cryoinjuries in post-thawed oocytes and embryos. Slow freezing with programmable freezers has the advantage of using low concentrations of cryoprotectants, which are usually associated with chemical toxicity and osmotic shock, but their ability to avoid ice-crystal formation at low concentrations is limited. Slow freezing also induces supercooling effects that must be avoided using manual or automatic seeding 4. In the vitrification process, high concentrations of cryoprotectants inhibit the formation of ice-crystals and lead to the formation of a glasslike vitrified state in which water is solidified, but not expanded. However, due to the toxicity of cyroprotectants at the concentrations used, oocytes/embryos can only be exposed to the cryoprotectant solution for a very short period of time and in a minimum volume solution, before submerging the samples directly in liquid nitrogen 5. In the last decade, vitrification has become more popular because it is a very quick method in which no expensive equipment (programmable freezer) is required. However, slow freezing continues to be the most widely used method for oocyte/embryo cryopreservation. In this video-article we show, step-by-step, how to collect and slowly freeze hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

In this video-article we present a step-by-step demonstration on how to collect and cryopreserve hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

Sammlung und Kryokonservierung von Oozyten Hamster und Maus Embryonen

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

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

Ein High-Throughput-Methode für Zebrabärblinge Sperma Kryokonservierung und In Vitro Fertilisation

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

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.

Kryokonservierung von Maus Embryonen durch Ethylen-Glykol-Based Vitrification

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

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generation der Mäuse aus induzierten pluripotenten Stammzellen

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Fish Sperm Assessment Using Software and Cooling Devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

Fisch Sperma Bewertung mit Software und Kühlgeräte

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...