Name: utero-tubal embryo transfer and vasectomy in the mouse model
Uploaded: 2014-02-28T13:45:00
Description: Watch this Scientific Journal Video about Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model at JoVE.com

Diese Arbeit wurde durch Mittel aus der Abteilung für Tier-und Vogel Wissenschaften BT unterstützt.

Alle Tierversuche wurden von der Beltsville Bereich Animal Care und Verwenden Gremien (BAACUC 11-015) in Übereinstimmung mit USDA Animal Care und Verwenden Richtlinien genehmigt. 1. Anästhesie und Analgesie (gemeinsam für beide Chirurgische Verfahren) <ol><li> Wiegen Sie die Maus, und laden Sie die folgenden Anästhetika und analgetische in zwei 1 ml Spritzen mit 27 G Nadeln: <ol><li> Ketamin (0,1 mg / g: 0,01 ml / g einer 10 mg / ml) und Xylazin (0,01 ...

<ol>
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Biol. 33, 394-416 (1956).</li><li>Goto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fate+of+embryos+transferred+into+the+uterus.">The fate of embryos transferred into the uterus.</a> J. Assist. Reprod. Gen. 10, 197-201 (1993).</li><li>Nagy, A., Gertsenstein, M., Vintersten, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the Mouse Embryo: A Laboratory Manual. , (2003).</li><li>Chin, H. J., Wang, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utero-tubal+transfer+of+mouse+embryos.">Utero-tubal transfer of mouse embryos.</a> Genesis. 30, 77-81 (2001).</li><li>Ramirez, M. A., Fernandez-Gonzalez, R., Perez-Crespo, M., Pericuesta, E., Gutierrez-Adan, A. <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+stem+cell+activation,+culture+media+of+manipulated+embryos,+and+site+of+embryo+transfer+in+the+production+of+F0+embryonic+stem+cell+mice.">Effect of stem cell activation, culture media of manipulated embryos, and site of embryo transfer in the production of F0 embryonic stem cell mice.</a> Biol. Reprod. 80, 1216-1222 (2009).</li><li>Miller, A. M., Wright-Williams, S. L., Flecknell, P. A., Roughan, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+abdominal+and+scrotal+approach+methods+of+vasectomy+and+the+influence+of+analgesic+treatment+in+laboratory+mice.">A comparison of abdominal and scrotal approach methods of vasectomy and the influence of analgesic treatment in laboratory mice.</a> Lab. Anim. 46, 304-310 (2012).</li><li>Flecknell, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory Animal Anaesthesia. , (2009).</li><li>Erhardt, W., Hebestedt, A., Aschenbrenner, G., Pichotka, B., Blumel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+with+various+anesthetics+in+mice+(pentobarbitone+ketamine-xylazine,carfentanyl-etomidate).">A comparative study with various anesthetics in mice (pentobarbitone ketamine-xylazine,carfentanyl-etomidate).</a> Res. Exp. Med. 184, 159-169 (1984).</li><li>Tarin, D., Sturdee, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+anaesthesia+of+mice:+evaluation+of+tribromo-ethanol,+ether,+halothane+and+methoxyflurane+and+development+of+a+reliable+technique.">Surgical anaesthesia of mice: evaluation of tribromo-ethanol, ether, halothane and methoxyflurane and development of a reliable technique.</a> Lab. Anim. 6, 79-84 (1972).</li><li>Zeller, W., Meier, G., Burki, K., Panoussis, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adverse+effects+of+tribromoethanol+as+used+in+the+production+of+transgenic+mice.">Adverse effects of tribromoethanol as used in the production of transgenic mice.</a> Lab. Anim. 32, 407-413 (1998).</li><li>Lieggi, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+and+safety+of+stored+and+newly+prepared+tribromoethanol+in+ICR+mice.">Efficacy and safety of stored and newly prepared tribromoethanol in ICR mice.</a> Contemp. Top. Lab. Anim. Sci. 44, 17-22 (2005).</li><li>Lieggi, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+preparation+methods+and+storage+conditions+of+tribromoethanol.">An evaluation of preparation methods and storage conditions of tribromoethanol.</a> Contemp. Top. Lab. Anim. Sci. 44, 11-16 (2005).</li><li>Meyer, R. E., Fish, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+tribromoethanol+anesthesia+for+production+of+genetically+engineered+mice+and+rats.">A review of tribromoethanol anesthesia for production of genetically engineered mice and rats.</a> Lab. Anim. 34, 47-52 (2005).</li><li>Chatot, C. L., Lewis, J. L., Torres, I., Ziomek, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+1-cell+embryos+from+different+strains+of+mice+in+CZB+medium.">Development of 1-cell embryos from different strains of mice in CZB medium.</a> Biol. Reprod. 42, 432-440 (1990).</li><li>Quinn, P., Barros, C., Whittingham, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+of+hamster+oocytes+to+assay+the+fertilizing+capacity+of+human+spermatozoa.">Preservation of hamster oocytes to assay the fertilizing capacity of human spermatozoa.</a> J. Reprod. Fertil. 66, 161-168 (1982).</li><li>Dios Hourcade, d. e., Perez-Crespo, J., Serrano, M., Gutierrez-Adan, A., A, B., Pintado, <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+and+in+vivo+development+of+mice+morulae+after+storage+in+non-frozen+conditions.">In vitro and in vivo development of mice morulae after storage in non-frozen conditions.</a> Reprod. Biol. Endocrinol. 10, 62 (2012).</li></ol>

Die Vasektomie ist eine relativ einfach Operationstechnik, die keine großen Schwierigkeiten verbunden ist. Wenn Desinfektion mit Povidon-Jod und Ethanol stellen Sie sicher, dass der letzte Waschanlage (mit Ethanol) entfernt Povidoniod, da es das Bauchfell reizen. Der Zugang zu Samenleiter kann auch durch den Hodensack oder Durchführen einer transversalen Schnitt in den Bauch 8 erreicht werden. Scrotal Einschnitt wurde empfohlen, Bauchschnitt durch den vergleichsweise kleineren Schnitt benötigt und...

Embryotransfer ist wahrscheinlich der häufigste chirurgische Eingriff im Mausmodell durchgeführt. Diese Technik ist wesentlich, Nachkommen von Embryonen in vitro Manipulation Techniken unterworfen wurde, und daher stellt ein notwendiger Schritt für die Entwicklung von genetisch modifizierten Modelle von Vorkern-Injektion lentiviralen Transduktion oder Chimärenbildung. Außerdem ermöglicht die Technik die Untersuchung der Auswirkungen auf die Entwicklung von diversen Beleidigungen während der Präimplantations-Entwicklung auftreten. Die Verwendung von künstlichen Reproduktionstechniken 1 oder der Einwirkung von abnormalen Konzentrationen verschiedener Stoffe oder Stoffwechsel 2. Mai Embryonalentwicklung was Implantation oder Plazenta Ausfälle und langfristigen Auswirkungen bei den Nachkommen beeinflussen. Eine zuverlässige und reproduzierbare Embryo-Transfer-Technik ist entscheidend, um die möglichen negativen Auswirkungen der experimentellen Behandlung bei der Implantation und der fetalen Entwicklung in einem konsistenten Menschen testenner. Murine Präimplantationsembryonen auf einen weiblichen Empfänger entweder in den Eileiter über den Ampullen von 0,5 Tage nach coitum (DPC) pseudo-Empfänger (Eileiter-Transfer) oder 3,4 in die Gebärmutter von 2,5 dpc schein Empfänger (Gebärmutter-Transfer) 5,6 übertragen werden je nach Entwicklungsstadium. Embryonen im Blastozystenstadium, wie sie verwendet werden, um chimäre Mäuse zu erzeugen, durch Injektion von embryonalen oder induzierte pluripotente Stammzellen, sind in der Regel durch die Uterus Übertragung übertragen. Blastozysten können auch in den Eileiter einer 0,5 dpc Empfänger übertragen werden, aber es stellt eine weniger physiologischen Test für Entwicklungs Disruptoren, weil der Embryo durchläuft Diapause und hat 2 Tage, um von der Beleidigung zu erholen, bevor die Implantation stattfindet. Gebärmutter Übertragung beinhaltet Stechen der Uteruswand mit einem schmalen Nadel, um eine Öffnung, die den Zugang eines Embryo-Pipette in das Uteruslumen ermöglicht erzeugen. Abwohl diese Technik gute Ergebnisse (dh der Anteil der übertragenen Embryonen, die zu einem Welpen entwickeln) ist das Überleben zu Begriff oft niedrig und unberechenbar 7,8. Die Punktion der Uteruswand bringt einige nachteilige Nebenwirkungen. Erstens ist Myometrium ein stark vaskularisierten Gewebes und seine Punktion führt oft zu einer geringen Blutung. Blut kann die Embryotransfer Pipette blockieren oder dringen in die Uteruslumen verursacht embryonalen Tod und / oder Implantation Versagen. Dies ist besonders relevant, wenn Embryonen ohne zona übertragen werden, da die Blutzellen und Ablagerungen können zu den Blastomeren befestigen. Zweitens ist die Öffnung nicht verschließen durchgeführt, nachdem die Embryonen übertragen wurden, so dass sie durch die Öffnung fließen kann und zurück in die Bauchhöhle ausgestoßen wird, wenn ein zu großes Volumen hat, in den Uterus eingeführt worden ist. Die beschriebene utero-Eileiter-Embryo-Transfer hier die Vorteile der utero-Eileiter-Kreuzung, um die embr liefernYos in den Uterus ohne die Notwendigkeit der Punktion der Uteruswand und damit die Vermeidung ihrer nachteiligen Folgen 9. Die pseudo-Empfänger für Frauen Embryotransfer verwendet werden, durch natürliche Paarung mit Männchen vasektomierten 8 erhalten. Die Samensekret durch eine sterile Männchen produziert werden für die Gebärmutter erforderlich empfänglich für die Embryonen übertragen zu werden. Um einen Empfänger zu erhalten, werden maximal 2 Weibchen von 8 Wochen bis 6 Monaten mit einem männlichen vasektomierten in den Nachmittag gelegt. Am nächsten Morgen sind Frauen für das Vorhandensein einer vaginalen Kopulation Stecker, einem Klumpen geronnenen Proteinen aus der männlichen Samenflüssigkeit überprüft. Als während der Mitternachts Paarung erfolgt in der Regel, wird der Tag der vaginalen Plug-Erkennung als 0,5 dpc sein. Obwohl vasektomierten Männer können von einigen Händlern erworben werden kann, ist die hier beschriebene chirurgische Verfahren relativ einfach und erfordert keine zusätzliche Instrumente als für Embryotransfer erforderlich.

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		<tr height="29">
			<td height="29" style="height:29px;width:232px;">Ketamine</td>
			<td style="width:111px;">VEDCO</td>
			<td style="width:203px;">Ketaved ANADA 200-257</td>
			<td style="width:691px;">To be ordered by a licensed veterinarian.</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:232px;">Xylazine</td>
			<td style="width:111px;">Lloyd laboratories</td>
			<td style="width:203px;">Anased NADA #139-236</td>
			<td style="width:691px;">To be ordered by a licensed veterinarian.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:232px;">Buprenorphine</td>
			<td style="width:111px;">Generic</td>
			<td style="width:203px;">NDC 400-42-010-01</td>
			<td style="width:691px;">To be ordered by a licensed veterinarian.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Eye ointment</td>
			<td style="width:111px;">Novartis</td>
			<td style="width:203px;">Genteal</td>
			<td style="width:691px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:232px;">Antibiotic</td>
			<td style="width:111px;">Pfizer</td>
			<td style="width:203px;">Clavamox NADA #55-101.</td>
			<td style="width:691px;">Added to drinking water (0.3 mg/ml of amoxicillin trihydrate and 0.075 mg/ml of clavulanate potassium). Add 1.5 ml of the reconstituted 15 ml bottle to 250 ml of water.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Dressing serrated forceps</td>
			<td style="width:111px;">ROBOZ</td>
			<td style="width:203px;">RS-8120</td>
			<td style="width:691px;">Any medium size surgical-grade steel straight forceps will work.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Micro dissecting serrated forceps</td>
			<td style="width:111px;">ROBOZ</td>
			<td style="width:203px;">RS-5137</td>
			<td style="width:691px;">These ones are curved at a 90&#186; angle. Straight forceps can be used if preferred.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Slight curved micro dissection forceps</td>
			<td style="width:111px;">ROBOZ</td>
			<td style="width:203px;">RS-5136</td>
			<td style="width:691px;">This model is particularly useful to hold the oviduct.</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:232px;">Scissors</td>
			<td style="width:111px;">ROBOZ</td>
			<td style="width:203px;">RS-5880</td>
			<td style="width:691px;">Any regular surgical grade steel small straight scissors will work.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">27G needles</td>
			<td style="width:111px;">Beckton-Dickinson</td>
			<td style="width:203px;">305136</td>
			<td style="width:691px;">Smaller needles (30G) can be also used. 25G may be a bit too big.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:232px;">Clip applier</td>
			<td style="width:111px;">MiKRon</td>
			<td style="width:203px;">42763</td>
			<td style="width:691px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:232px;">9 mm Clips</td>
			<td style="width:111px;">MiKRon</td>
			<td style="width:203px;">427631</td>
			<td style="width:691px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Clip remover</td>
			<td style="width:111px;">MiKRon</td>
			<td style="width:203px;">7637</td>
			<td style="width:691px;">Two pairs of teeth forceps (ROBOZ RS-8160) can be used instead.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:232px;">Suture needle holder</td>
			<td style="width:111px;">ROBOZ</td>
			<td style="width:203px;">RS-7820</td>
			<td style="width:691px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:232px;">Suture</td>
			<td style="width:111px;">Dowist Gell</td>
			<td style="width:203px;">5-0 Dexon S 7204-21</td>
			<td style="width:691px;">Can be substituted for any 4-0 to 6-0 absorbable suture with a narrow curved needle.</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:232px;">Glass capillaries</td>
			<td style="width:111px;">VWR</td>
			<td style="width:203px;">100 ul calibrated pipettes 53432-921</td>
			<td style="width:691px;">It includes a mouth aspirator system that only requires to attach a 0.22 um filter in the tubing to be ready to use. More information can be obtained in ref. 8.</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:232px;">Burner</td>
			<td style="width:111px;">KISAG AG</td>
			<td style="width:203px;">Typ 2002</td>
			<td style="width:691px;">Gas operated burner, can be charged with Kigas (CH-4512, from the same vendor). Alcohol burners may be also used, but gas provides a higher temperature and this burner provides a small and precise flame.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:232px;">Stereomicroscope</td>
			<td style="width:111px;">Leica</td>
			<td style="width:203px;">MZFLIII</td>
			<td style="width:691px;">This is an expensive estereomicroscope with fluorescence, that can be also used for other purposes. There are cheaper options such as Leica MZ8 or Nikon SMZ-10 or SMZ-2B, to name a few. It is better to use two stereomicroscopes, one for handling the embryos (which does not need to be a very nice one) and another one for the recipient. The one used for the recipient should display a long distance from the stage plate to the objective lense, in order to be able to focus 3-4 cm above the stage plate (where the oviduct will be placed) and still leave some room for the surgeon; most of the stereomicroscopes can do this, but some cannot.&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:232px;">Fiber optics ilumination</td>
			<td style="width:111px;">Dolan Jenner</td>
			<td style="width:203px;">Fiber lite</td>
			<td style="width:691px;">To iluminate the surgical area. There are different systems available.</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:232px;">Warm stages</td>
			<td style="width:111px;">American scope</td>
			<td style="width:203px;"><a href="https://exch.mail.umd.edu/owa/redir.aspx?C=1qTJKejamUSfudeYG_MlZHvTY3dsJM9Iaqr2SnwGD4ECWGzB8e8Rpg--_L7u0vJmyatXQJzGQ0k.&amp;URL=http%3a%2f%2fstore.amscope.com%2ftcs-100.html" target="_blank">http://store.amscope.com/tcs-100.html</a></td>
			<td style="width:691px;">These can be placed over the stage plate of the stereomicroscope. Some modifications (inserting a stick to level the stage) may be needed if it is too short for the stereomicroscope. A big warm stage can be used for warming the cage if it is available. If not, a regular heating pad can be used, but temperature must be checked.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Culture dishes for embryo manipulation</td>
			<td style="width:111px;">Falcon</td>
			<td style="width:203px;">353001</td>
			<td style="width:691px;">351008 may be also used, they made narrower drops.</td>
		</tr>
	</tbody>
</table>

utero-tubal embryo transfer and vasectomy in the mouse model

Die Übertragung der Präimplantationsembryonen zu einem Surrogat Frau ist ein erforderlicher Schritt für die Herstellung von genetisch veränderten Mäusen, um zu studieren oder die Auswirkungen von epigenetischen Veränderungen während Präimplantations Entwicklung bei zukünftigen Entwicklung des Fötus und Erwachsene Gesundheit entstanden ist. Die Verwendung einer wirksamen und kohärenten Embryo-Transfer-Technik ist entscheidend, um die Erzeugung von gentechnisch veränderten Tieren zu verbessern und die Wirkung der verschiedenen Behandlungen auf Implantationsraten und Überlebenszeit zu bestimmen. Embryos im Blastozysten-Stadium in der Regel durch Uterus Übertragung übertragen, Ausführen einer Punktion in der Uteruswand, die Embryomanipulation Pipette einzuführen. Die in der Gebärmutter durchgeführt Öffnung nicht schließen, nachdem die Pipette wurde zurückgezogen, und die Embryonen können Abfluss in die Bauchhöhle durch den Überdruck der Gebärmutter. Die Punktion kann auch produzieren eine Blutung, die Implantation beeinträchtigt, blockiert die Transferpipette und können Embryo d beeinflussenntwicklung, vor allem, wenn Embryonen ohne zona übertragen werden. Folglich ist diese Technik oft zu sehr variabel und insgesamt niedrigen Embryo-Überlebensraten. Die Vermeidung dieser negativen Auswirkungen, Eileiter-utero-Embryo-Transfer nutzen die utero-Eileiter-Kreuzung als eine natürliche Barriere, die Embryo Abfluss behindert und vermeiden Sie die Punktion der Gebärmutterwand. Vasektomierten Männer werden für den Erhalt schein Empfänger erforderlich. Eine Technik zur Vasektomie durchführen wird als Ergänzung zu den utero-Eileiter-Embryo-Transfer beschrieben.

Utero-Eileiter-Embryo-Transfer bietet eine mittlere Embryonen in die Gebärmutter vermeiden einige der Komplikationen an der Gebärmutterembryotransfer 2,9,10 verbunden zu übertragen. In Tabelle 1 zeigen wir einige repräsentative Ergebnis, das wir erhalten haben, die Übertragung CD1 Blastozysten, verschiedene Arten von Manipulationen CD1 Empfänger unterzogen nach dem beschriebenen Protokoll. Das Überleben zu Begriff (% der Embryonen, was zu einer Welpe) oder das Überleben bis E15 (im Fa...

Watch this Scientific Journal Video about Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model at JoVE.com

Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model

Utero-Eileiter-Embryo-Transfer verwendet die utero-Eileiter-Kreuzung als Barriere, um den Embryo Abfluss, die bei der Durchführung von Uterus Übertragung auftreten können, zu verhindern. Vasektomierten Männchen sind erforderlich, um pseudo-Empfänger für Embryotransfer erhalten. Beide Techniken werden diskutiert.

Utero-Tubenembryotransfer und Vasektomie im Maus-Modell

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects ...

utero-tubal-embryo-transfer-and-vasectomy-in-the-mouse-model

Research

JoVE Journal

Biology

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.

Nuclear Transfer in Maus-Oozyten

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

Forschung

Biologie

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

Assay für Neural Induction im Hühnerembryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

Elektrophysiologische Aufzeichnung in der Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

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.

In-vivo-Imaging und therapeutische Behandlungen in einem orthotopen Mausmodell des Eierstockkrebs

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

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

Tracking Morphogenetic Tissue Verformungen in der frühen Hühnerembryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

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 ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Maus embryonalen Entwicklung in einem Serum-freien Whole Embryo Culture-System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

Validierung eines Maus-Modell zu LINC Komplexe Disrupt in einem zellspezifisch

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.

Zellkernisolierung aus der Niere adulter Mäuse für die Einzelkern-RNA-Sequenzierung

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are ...

Durch zervikale Manipulation kam es zu einer Pseudoschwangerschaft bei Mäusen für die assistierte Reproduktion

Inducing Pseudopregnancy in Female Mice Without the Need for Vasectomized Males Prior to Non-Surgical Embryo Transfer or Artificial Insemination

For successfully maintaining pregnancy with embryo transfer or artificial insemination,&#160;female recipient mice must be induced into a pseudopregnant state. Female mice are traditionally paired overnight with vasectomized males, and the following morning, the presence of a copulation plug is assessed. To increase the efficiency of producing pseudopregnant females, a cervical manipulation technique has been standardized to be used in combination with non-surgical embryo transfer or artificial insemination techniques in mice. The blunt end of a small plastic rod is inserted vaginally to contact the cervix and is vibrated for 30 s by contact with a trimmer. The procedure is quick and does not require anesthesia or analgesia. This technique increases the reliability and predictability of producing pseudopregnant females and entirely eliminates the requirement for vasectomized males. For CD1 mice, the efficiency of pseudopregnancy induction using cervical manipulation was 83% for females in estrus (N = 76) but only 38% of females in estrus were plugged by vasectomized males (N = 24). Artificial insemination in CD1 mice was performed by estrus synchronization with hormones, cervical manipulation, and the uterine transfer of sperm. Artificial insemination recipients receiving cervical manipulation (N = 76) had a pregnancy rate of 72% and an average litter size of 8.3 pups. This method can also be used to produce pseudopregnant females for non-surgical embryo transfer. Therefore, inducing pseudopregnancy by cervical manipulation is a convenient and efficient alternative to mating with a vasectomized male when performing non-surgical assisted reproduction techniques. Using cervical manipulation provides 3Rs (replacement, reduction, and refinement) benefits for assisted reproduction techniques by reducing the number of animals required and eliminating the necessity for surgically altered males.

Autor im Rampenlicht: Herbeiführung einer Pseudoschwangerschaft bei weiblichen Mäusen ohne die Notwendigkeit vasektomierter Männchen vor einem nicht-chirurgischen Embryotransfer oder einer künstlichen

The cervical manipulation method presented can induce pseudopregnancy in mice without the need for breeding females with vasectomized males. The induction of pseudopregnancy is required for the success of non-surgical embryo transfer and non-surgical artificial insemination, both of which are also presented.

Induktion einer Pseudoschwangerschaft bei weiblichen Mäusen, ohne dass vasektomierte Männchen vor einem nicht-chirurgischen Embryotransfer oder einer künstlichen Befruchtung erforderlich sind

For successfully maintaining pregnancy with embryo transfer or artificial insemination,&#160;female recipient mice must be induced into a pseudopregnant ...