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

Este trabajo fue apoyado por fondos del Departamento de Ciencia Animal y de aves de BT.

Todos los experimentos con animales fueron aprobadas por el Cuidado de Animales y el empleo Comites Área Beltsville (BAACUC 11-015), de acuerdo con el USDA y Cuidado de Animales Pautas del uso. 1. Anestesia y Analgesia (comunes a los procedimientos quirúrgicos) <ol><li> Pesar el ratón y cargar los siguientes anestésicos y analgésicos en dos jeringas de 1 ml con 27 g de agujas: <ol><li> La ketamina (0,1 mg / g: 0,01 ml / g de una solución de 10 mg / m...

<ol>
	<li>Fernandez-Gonzalez, R., 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=Long-term+effect+of+in+vitro+culture+of+mouse+embryos+with+serum+on+mRNA+expression+of+imprinting+genes,+development,+and+behavior.">Long-term effect of in vitro culture of mouse embryos with serum on mRNA expression of imprinting genes, development, and behavior.</a> Proc. Natl. Acad. Sci USA. 101, 5880-5885 (2004).</li><li>Bermejo-Alvarez, P., Roberts, R. M., Rosenfeld, C. S. <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+glucose+concentration+during+in+vitro+culture+of+mouse+embryos+on+development+to+blastocyst,+success+of+embryo+transfer,+and+litter+sex+ratio.">Effect of glucose concentration during in vitro culture of mouse embryos on development to blastocyst, success of embryo transfer, and litter sex ratio.</a> 79, 329-336 (2012).</li><li>Tarkowski, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experiments+on+the+development+of+isolated+blastomers+of+mouse+eggs.">Experiments on the development of isolated blastomers of mouse eggs.</a> Nature. 184, 1286-1287 (1959).</li><li>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=Fertilization+of+mouse+eggs+in+vitro.">Fertilization of mouse eggs in vitro.</a> Nature. 220, 592-593 (1968).</li><li>McLaren, A., Biggers, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+development+and+birth+of+mice+cultivated+in+vitro+as+early+as+early+embryos.">Successful development and birth of mice cultivated in vitro as early as early embryos.</a> Nature. 182, 877-878 (1958).</li><li>McLaren, A., Michie, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+transfer+of+fertilized+mouse+eggs+to+uterine+foster-mothers.+I.+Factors+affecting+the+implantation+and+survival+of+native+and+transferred+eggs.">Studies on the transfer of fertilized mouse eggs to uterine foster-mothers. I. Factors affecting the implantation and survival of native and transferred eggs.</a> J. Exp. 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>

La vasectomía es una técnica quirúrgica relativamente sencillo que no implica mayores dificultades. Cuando desinfecte con yodo povidona y etanol asegúrese de que el último lavado (con etanol) elimina la povidona yodada, ya que puede irritar el peritoneo. El acceso a los conductos deferentes también se puede lograr por el escroto o la realización de una incisión transversal en el abdomen 8. Incisión escrotal ha sido recomendado para transversales incisión abdominal debido a la incisión rela...

La transferencia de embriones es probablemente el procedimiento quirúrgico más frecuente se realiza en el modelo de ratón. Esta técnica es esencial para obtener la descendencia a partir de embriones sometidos a técnicas de manipulación in vitro en y, por lo tanto, constituye un paso necesario para el desarrollo de modelos modificados genéticamente mediante inyección pronuclear, la transducción lentiviral, o formación de quimeras. Además, la técnica permite el estudio de los efectos sobre el desarrollo de los diversos insultos que ocurren durante el desarrollo de preimplantación. El uso de técnicas de reproducción artificial 1 o la exposición a concentraciones anormales de diferentes sustancias o metabolitos 02 de mayo afectar el desarrollo del embrión que resulta en la implantación o placentación fallos y efectos a largo plazo en la descendencia. Una técnica de transferencia de embriones fiable y reproducible es crucial para probar los posibles efectos negativos del tratamiento experimental en la implantación y el desarrollo fetal en un hombre coherentener. Preimplantación de embriones murinos se pueden transferir a una hembra receptora, ya sea en el oviducto a través de las ampollas de 0,5 días postcoital (dpc) receptores pseudopreñadas (de transferencia) 3,4 oviducto o al útero de 2,5 dpc pseudopreñada receptor (transferencia uterina) 5,6 dependiendo de su estado de desarrollo. Los embriones en la etapa de blastocisto, tales como los que se utilizan para generar ratones quiméricos mediante la inyección de células madre pluripotentes embrionarias o inducidas, se transfieren generalmente por transferencia uterina. Los blastocistos pueden también ser transferidos al oviducto de un DPC receptor 0.5, pero constituye una prueba menos fisiológica para los disruptores de desarrollo, debido a que el embrión se somete a diapausa y tiene 2 días para recuperarse de la lesión antes de la implantación tiene lugar. Transferencia uterina implica la punción de la pared uterina con una aguja estrecha con el fin de generar una abertura que permite el acceso de una pipeta de manipulación de embrión en el lumen uterino. Launque esta técnica puede dar buenos resultados, la supervivencia a plazo (es decir, el porcentaje de embriones transferidos que se desarrollan a un cachorro) suele ser baja e impredecible 7,8. La perforación de la pared uterina conlleva algunos efectos secundarios perjudiciales. En primer lugar, miometrio es un tejido altamente vascularizado y su punción a menudo resulta en una pequeña hemorragia. La sangre puede bloquear la pipeta de transferencia de embriones o invadir el lumen uterino causando la muerte embrionaria y / o fallo de implantación. Esto es particularmente relevante cuando los embriones se transfieren sin zona, ya que las células de la sangre y los desechos pueden adherirse a los blastómeros. En segundo lugar, la apertura realizada no sella después de que los embriones se han transferido, por lo que puede fluir de vuelta a través del orificio y ser expulsado a la cavidad abdominal cuando un volumen demasiado grande ha sido introducir en el útero. La transferencia de embriones útero-tubárica se describe en el presente documento se aprovechan de la unión útero-tubárica para entregar el EMBRyos en el útero sin la necesidad de perforar la pared uterina y de ese modo evitar sus consecuencias adversas 9. Las hembras receptoras pseudopreñadas utilizados para la transferencia de embriones se obtienen mediante monta natural con machos vasectomizados 8. Las secreciones seminales producidos por un macho estéril son necesarios para el útero a ser receptivo a los embriones transferidos. Para obtener un receptor, un máximo de 2 hembras de 8 semanas a 6 meses de edad se colocan con un hombre sometido a la vasectomía en la tarde. A la mañana siguiente, las hembras se comprueba la presencia de un tapón de la cópula vaginal, un grupo de proteínas coaguladas del líquido seminal masculino. Como el apareamiento ocurre generalmente durante la medianoche, el día de la detección de tapón vaginal se considera 0,5 dpc. Aunque los machos vasectomizados se pueden adquirir en algunos vendedores, el procedimiento quirúrgico descrito en el presente documento es relativamente fácil y no requiere ningún instrumentos adicionales a los requeridos para la transferencia de embriones.

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	<tbody>
		<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

La transferencia de los embriones de preimplantación a una hembra sustituto es un paso necesario para la producción de ratones modificados genéticamente o para estudiar los efectos de las alteraciones epigenéticas originadas durante el desarrollo de preimplantación en la posterior desarrollo y adulto fetal salud. El uso de una técnica de transferencia de embriones eficaz y coherente es crucial para mejorar la generación de animales modificados genéticamente y para determinar el efecto de diferentes tratamientos en las tasas de implantación y la supervivencia a largo plazo. Los embriones en la etapa de blastocisto se transfieren generalmente por transferencia uterina, la realización de una punción en la pared uterina para introducir la pipeta de manipulación de embriones. El orificio realizado en el útero no se cierra después de que la pipeta ha sido retirada, y los embriones puede salida a la cavidad abdominal debido a la presión positiva del útero. La punción también puede producir una hemorragia que afecta la implantación, bloquea la pipeta de transferencia y puede afectar el embrión desarrollo, especialmente cuando se transfieren los embriones sin zona. En consecuencia, esta técnica a menudo resulta en tasas de supervivencia bajas embrión muy variables y globales. Evitar estos efectos negativos, la transferencia de embriones útero-tubárica tomar ventaja de la unión útero-tubárica como una barrera natural que impide la salida del embrión y evitar la punción de la pared uterina. Se requieren machos vasectomizados para la obtención de los receptores pseudopreñadas. Una técnica para llevar a cabo la vasectomía se describe como un complemento a la transferencia de embriones útero-tubárica.

Transferencia de embriones útero-tubárica proporciona un medio para transferir los embriones al útero para evitar algunas de las complicaciones asociadas a la transferencia de embriones del útero 2,9,10. En la Tabla 1 se muestra algún resultado representante hemos obtenido la transferencia de blastocistos CD1 sometidas a diferentes tipos de manipulaciones a destinatarios CD1 siguiendo el protocolo descrito. La supervivencia a plazo (% de los embriones resultantes en un cachorro) o la sup...

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

La transferencia de embriones útero-tubárica utiliza la unión útero-tubárica como una barrera para impedir la salida de embriones que puede ocurrir cuando se realiza la transferencia uterina. Se requieren machos vasectomizados obtener destinatarios pseudopreñadas para la transferencia de embriones. Ambas técnicas se discuten.

Transferencia de Embriones Utero-de trompas y vasectomía en el modelo de ratón

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.

Transferencia nuclear en ovocitos de ratón

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

Investigación

Biología

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.

Ensayo de inducción neural en el embrión de pollo

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.

Registro electrofisiológico en el Drosophila Embrión

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.

En vivo de imágenes y tratamientos terapéuticos en un modelo de ratón ortotópico de cáncer de ovario

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.

El seguimiento de deformaciones morfogenética del tejido en el embrión de pollo temprano

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.

Generación de ratones derivados de células madre pluripotentes inducidas

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.

Embriones de ratón con el desarrollo en todo un sistema de cultivo de embriones sin suero

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.

Validación de un modelo de ratón para interrumpir LINC Complejos en una celda de manera específica

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.

Aislamiento de núcleos de riñón de ratón adulto para secuenciación de ARN de un solo núcleo

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

Manipulación cervical inducida por pseudoembarazo en ratones para reproducción asistida

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 destacado: Inducir pseudoembarazo en ratones hembra sin necesidad de machos vasectomizados antes de la transferencia embrionaria no quirúrgica o la inseminación artificial  

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.

Inducir pseudoembarazo en ratones hembra sin necesidad de machos vasectomizados antes de la transferencia embrionaria no quirúrgica o la inseminación artificial

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