Esta investigación fue apoyada [en parte] por el programa de investigación intramuros del NIH, Instituto Nacional de Ciencias de salud ambiental. Agradecimiento especial a Shyamal Peddada para el análisis estadístico. También queremos agradecer a Humphrey Yao, Gary Ave y Yuki Arao para la revisión de este manuscrito y Franco DeMayo por su continuo apoyo y asesoramiento.

Todas las operaciones se realizaron como parte del funcionamiento normal de la instalación de NIEHS Knock Out Mouse base. Todos los ratones fueron mantenidos en un 12:12 ciclo de h luz: oscuridad y se alimentan una dieta de agua y NIH-31 ad libitum. Todos los experimentos fueron realizados según el cuidado Animal y Comité de uso del nacional Instituto de Ciencias de salud ambiental.
1. animal preparación
<ol><li>Raza de ratones donantes, B6 (Cg)-Tyr < c-2J > ratones femeninos/j 8-10 semanas de edad naturalmente a B6 (Cg)-Tyr < c-2J > /J ratones machos entre 8 y 26 semanas de edad. Verificación se conecta visualmente a la mañana siguiente (E0.5).</li>
	<li>Se crían ratones receptores, Swiss Webster ratones hembra entre 6 y 9 semanas de edad se crían naturalmente a vasectomía ratones machos Swiss Webster. Verificación se conecta visualmente a la mañana siguiente (E0.5).</li>
</ol>2. ES la célula preparación
<ol><li>Para derieved 129 AB2.2 ES cells, cultura células en DMEM suplementado con 15% FBS, 1% glutamina, 1% Penn/strep y 0.1% β-mercaptoetanol. Cultura todas las células de otras ES siguiendo las instrucciones del proveedor de celular ES, utilizando los protocolos recomendados.</li>
</ol>3. embrión colección
<ol><li>
Disección de animales
	<ol>
<li>Preparar varios platos de 60 mm, aproximadamente 1 por 5 ratones más suplemento 3, con 10 mL ráfaga colección los medios de comunicación (BCM) (10% FBS en DMEM), y un 4 plato bien con 0.5 mL de BCM por pozo. Equilibrar a 37 ° C, 5% CO2.</li>
		<li>Eutanasia a B6 (Cg)-Tyr < c-2J > ratones femeninos /J en E3.5 CO2 asfixia, con un caudal de 10%.</li>
		<li>Después euthanizing, coloque el ratón en la posición supina y mojado el abdomen con etanol al 70%.</li>
		<li>Hacer la primera incisión a través de la piel, en el punto medio del abdomen, haciendo la apertura tan amplia como sea posible. A continuación abra a través de la pared abdominal, otra vez hacer la incisión lo más amplia posible. Nota: Hacerlo en dos pasos ayuda a reducir o eliminar la contaminación de la piel.</li>
		<li>A partir de un ovario y sostiene la almohadilla grasa del ovario con fórceps romos, retire con cuidado el ovario, oviducto y útero, teniendo mucho cuidado para mantener el tejido adiposo a un mínimo. Extirpar el útero como una sola unidad, o como los cuernos, sin cambio en el procedimiento siguiente.</li>
		<li>Después del retiro, colocar el útero en un plato de 60 mm que contiene medios de colección ráfaga.</li>
	</ol>
</li>
	<li>
Eliminación de embriones
	<ol>
<li>Preparar una jeringa de 10 mL llenada de BCM y montar con una aguja de 25 G.</li>
		<li>Un útero a la vez, les quite el plato de sujeción de BCM, seque en un paño para quitar el exceso de sangre y coloque en un plato de 60 mm con un aumento bajo microscopio de disección.</li>
		<li>Sujetando con cuidado y suavemente del útero justo por debajo del extremo distal (cerca del oviducto) con pinzas de disección, inserte la aguja en el centro del útero en como paralelo un plano como sea posible. Insertar la aguja más allá de las puntas de las pinzas.</li>
		<li>Aplicar presión a las pinzas para sujetar el útero a la aguja y expulsar aproximadamente 1 mL de BCM a través del cuerno del útero. Nota: Debe tenerse cuidado en este paso, ya que demasiada presión sobre la jeringa causará un flujo incontrolable de los medios de comunicación, a menudo dejando el plato.</li>
		<li>Después del lavado del útero, útero entero hasta 5 por plato 60 mm, vuelva a colocar el plato en la incubadora hasta la colección.</li>
	</ol>
</li>
	<li>
Colección de embriones
	<ol>
<li>Utilizando una pipeta de cristal dibujado de la mano y un aparato de pipetas de boca, recoger blastocistos en un ámbito de disección, entre 25 X y 35 X. (Figura 1). Nota: Aparato de pipetas de boca consta de 30-40 cm de tubería flexible, menores de 5 mm de diámetro interior Inserte un 1 000 μl Punta de pipeta, punto primero en un extremo y colocar la pipeta de vidrio en él. Agregar un filtro de jeringa en el otro extremo, con un atado 1 cc jeringa barril, sin émbolo, a actuar como una pieza de la boca.</li>
		<li>Eliminar platos de la incubadora y usando un patrón cuadriculado, examinaron cada parte del plato para embriones. Embriones se recogen hasta individualmente y coloque sobre la gota de BCM en un plato secundario.</li>
		<li>Después de recoger de todos los platos, lavar los embriones una vez más en una gota de dulce BCM y luego colocar en el plato bien 4 a la espera de la inyección.</li>
	</ol>
</li>
</ol>4. embrión inyección
<ol><li>Recoger células madre embrionarias individualmente con la aguja de inyección basada en la confirmación.<ol>
<li>Preparar un plato de inyección mediante la adición de 5-7 mL de medio de inyección (DMEM, 10% FBS y 20 mM HEPES) a un plato de inyección de fondo de cristal. Las inyecciones se realizan a temperatura ambiente.</li>
		<li>Uso microinyector aire o aceite, aplique vacío a la aguja para extraer células madre embrionarias. Tenga cuidado de mantener como poco espacio como sea posible entre las células. Cuentan las células ES aquí o en la etapa de inyección.</li>
		<li>Seleccione las celdas ES que son de mediano a pequeño tamaño en comparación con la población en general de las células, con una superficie lisa y sin defectos obvios como las vacuolas. Nota: Poblaciones celulares ES varían mucho en calidad morfológica, y las normas utilizadas tendrán que ajustar con ellos.</li>
	</ol>
</li>
	<li>Inyectar los embriones con aproximadamente 15 células madre embrionarias (figura 1).<ol>
<li>Colocar una pipeta de sujeción punta un microinyector segundo de aire y coloque un embrión cerca de la abertura del titular. Se aplica vacío para sujetar el embrión al titular.</li>
		<li>Ajuste para el eje z moviendo la pipeta holding hacia arriba o hacia abajo para que el embrión apenas toca la diapositiva, seguida de concentración de la multa en la zona. A continuación, ajuste el eje Z de la aguja de inyección para que células madre embrionarias cerca de la punta están en foco.</li>
		<li>Ajuste el embrión ahora empujando suavemente de la aguja de inyección en la parte superior o inferior, girando para poner el ICM en la posición deseada.</li>
		<li>Inserte la aguja a través de la zona y el ICM o trofoblasto, usando una presión firme pero controlada. Aplicar una ligera presión a la microinyectora de la aguja de inyección para ayudar a mantener el blastocoel de derrumbarse.</li>
		<li>Una vez que la aguja ha penetrado el blastocoel, aplicar presión de la microinyectora para transferir células madre embrionarias para el blastocoel del embrión. PAUSE brevemente como el bisel de la aguja empieza a salir el embrión para permitir la presión en el embrión para volver a la normalidad, al tiempo que conserva las células. Nota: Referencia grupo células madre embrionarias fueron inyectados normalmente, siguiendo un tradicional ES la célula de inyección protocolo9, teniendo cuidado de no para tocar el ICM. También se inyectaron células madre embrionarias a través del ICM, empuja la aguja directamente a través de la ICM (TICM) (figura 2película suplementario).</li>
	</ol>
</li>
</ol>5. transferencia de eEmbryos
<ol><li>Transferencia de embriones de ratones Swiss Webster en E2.5, utilizando un dispositivo de transferencia embrionaria no quirúrgica, por procedimientos de fabricación. Este procedimiento no quirúrgico no requiere anestesia ni cuidados especiales después de la operación. Ratones de la casa individualmente después de la transferencia de embriones y a través de la entrega y posterior al destete.</li>
</ol>

<ol>
	<li>Cong, L., 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=Multiplex+Genome+Engineering+Using+CRISPR/Cas+Systems.">Multiplex Genome Engineering Using CRISPR/Cas Systems.</a> Science. 339 (6), 819-823 (2013).</li><li>Mali, P., 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=RNA-guided+human+genome+engineering+via+Cas9.">RNA-guided human genome engineering via Cas9.</a> Science. 339 (6), 823-826 (2013).</li><li>Friedel, R., Seisenberger, C., Kaloff, C., Wurst, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EUCOMM+the+European+Conditional+Mouse+Mutagenesis+Program.">EUCOMM the European Conditional Mouse Mutagenesis Program.</a> Brief Funct Genomic Proteomic. 6 (3), 180-185 (2007).</li><li>Austin, C. P., 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+knockout+mouse+project.">The knockout mouse project.</a> Nat Genet. 36 (9), 921-924 (2004).</li><li>Coleman, J. L., Brennan, K., Ngo, T., Balaji, P., Graham, R. M., Smith, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Knockout+and+Reporter+Mouse+Line+Generation+and+Breeding+Colony+Establishment+Using+EUCOMM+Conditional-Ready+Embryonic+Stem+Cells:+A+Case+Study.">Rapid Knockout and Reporter Mouse Line Generation and Breeding Colony Establishment Using EUCOMM Conditional-Ready Embryonic Stem Cells: A Case Study.</a> Front Endocrinol (Lausanne). 30, 105 (2015).</li><li>Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W., Roder, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+completely+cell+culture-derived+mice+from+early-passage+embryonic+stem+cells.">Derivation of completely cell culture-derived mice from early-passage embryonic stem cells.</a> Proc Natl Acad Sci U S A. 90 (18), 8424-8428 (1993).</li><li>Eggan, K., 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=Male+and+female+mice+derived+from+the+same+embryonic+stem+cell+clone+by+tetraploid+embryo+complementation.">Male and female mice derived from the same embryonic stem cell clone by tetraploid embryo complementation.</a> Nat Biotechnol. 20 (5), 455-459 (2002).</li><li>Eggan, K., 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=Hybrid+vigor,+fetal+overgrowth,+and+viability+of+mice+derived+by+nuclear+cloning+and+tetraploid+embryo+complementation.">Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation.</a> Proc Natl Acad Sci U S A. 98 (11), 6209-6214 (2001).</li><li>Eakin, G. S., Hadjantonakis, A. K., Papaioannou, V. E., Behringer, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+potential+and+behavior+of+tetraploid+cells+in+the+mouse+embryo.">Developmental potential and behavior of tetraploid cells in the mouse embryo.</a> Dev Biol. 288 (1), 150-159 (2005).</li><li>Huang, J., 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=Efficient+production+of+mice+from+embryonic+stem+cells+injected+into+four-+or+eight-cell+embryos+by+piezo+micromanipulation.">Efficient production of mice from embryonic stem cells injected into four- or eight-cell embryos by piezo micromanipulation.</a> Stem Cells. 26 (7), 1883-1890 (2008).</li><li>Poueymirou, W. T., 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=F0+generation+mice+fully+derived+from+gene-targeted+embryonic+stem+cells+allowing+immediate+phenotypic+analyses.">F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses.</a> Nat Biotechnol. 25 (1), 91-99 (2007).</li><li>Behringer, R., Gertsenstein, M., Nagy, K. V., Nagy, 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> Manipulating the Mouse embryo. , (2014).</li><li>Pease, S., Saunders, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advanced Protocols for Animal Transgenesis. , (2011).</li><li>Young, M. 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Los autores tienen intereses que compiten a revelar.

Durante mucho tiempo se ha pensado que cualquier disturbio del ICM podría conducir a la mortalidad, de hecho, los protocolos actuales de microinyección blastocisto todavía advierten de esta12,13. Lo que hemos mostrado aquí es la microinyección a través de la RIC no es perjudicial para el embrión y aumenta la producción de quimeras.
El mecanismo exacto de este efecto no ha sido identificado. Sin embargo, la interrupción mecáni...

Durante casi 25 años, la creación de ratones genéticamente apuntados ha sido un proceso lento, a menudo obstaculizado por un cuello de botella en la etapa de blastocistos ES células microinyección para la generación de germline transmisión de quimeras. Los avances recientes, incluyendo CRISPR/Cas91,2 en células madre embrionarias y de alto rendimiento ES célula consorcios de generación como KOMP, han mejorado la generación y disponibilidad de modificados genéticamente células madre embrionarias. Sin embargo, la generación de quimeras del germline sigue siendo un cuello de botella en la creación de ratones modificados genéticamente de estas células ES3,4. Proyectos de generación de alto rendimiento ES la célula han estado plagados de alta variabilidad en la calidad de células ES, que es crítica para la exitosa creación de quimeras del germline. Además, algunas de las líneas de células ES comúnmente utilizadas son conocidos por aneuploidia alta y la difícil producción de germline quimeras competentes5.
Muchos métodos se han desarrollado para la creación de ratones con una quimera o superior, o totalmente celular ES derivado ratones6,7,8,9,10. Mientras que cada uno de estos sistemas tiene sus propios méritos, también tienen sus defectos. La generación de quimeras tetraploides, mientras que la generación de celulares ES de 100% derivados de ratones, es ineficiente y generalmente limitado a líneas exogámica 5,6. Más nuevos métodos de inyección de mórula pueden producir alto porcentaje quimeras, cercana al 100%, pero generalmente requieren equipo adicional significativa (láser o piezoeléctricos) y capacitación10,11. En laboratorios donde estas técnicas ya están en marcha, esta nueva metodología puede no ser necesaria.
Objetivo de este estudio fue encontrar un método que utiliza técnicas comunes y equipos disponibles para aumentar la velocidad de generación de quimera, aumentando la posibilidad de transmisión del germline del alelo del mutante para establecer la colonia de ratón posterior.

<table><tbody><tr><td>ES Cell injection needle</td><td>Humagen</td><td>MSC-20-0</td><td></td></tr><tr><td>NSET device</td><td>Paratechs</td><td>60010</td><td></td></tr><tr><td>DMEM</td><td>Gibco (life technologies)</td><td>11965-092</td><td></td></tr><tr><td>FBS</td><td>Gibco (life technologies)</td><td>10439-024</td><td>lots should be individually tested for ES Cell compatibility.</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H0887</td><td></td></tr><tr><td>&amp;#946;-mercaptoethanol</td><td>Sigma</td><td>M7522</td><td></td></tr><tr><td>Micro manipulator</td><td>Leica</td><td>Micro manipulator</td><td></td></tr><tr><td>micro injector</td><td>Eppendorf AG</td><td>Cell Tram Air 5176</td><td></td></tr><tr><td>Microscope</td><td>Leica</td><td>DM IRB</td><td></td></tr><tr><td>4 well dish</td><td>Thermo Scientific</td><td>176740</td><td></td></tr><tr><td>60 mm dish</td><td>Sarstedt</td><td>83.3901</td><td></td></tr><tr><td>B6(Cg)-Tyr&amp;#60;c-2J&amp;#62;/J</td><td>Jackson Labs, Bar Harbor, Maine, USA</td><td>000058</td><td></td></tr><tr><td>Swiss Webster mice</td><td>Taconic, USA</td><td>Tac:SW</td><td></td></tr><tr><td>Injection Dish</td><td>MatTek Corp.</td><td>P50G-0-30-F</td><td></td></tr></tbody></table>

trans-inner cell mass injection of embryonic stem cells leads to higher chimerism rates

En un esfuerzo por aumentar la eficiencia en la creación de ratones modificados genéticamente a través de metodologías ES celular, presentamos una adaptación del protocolo actual de inyección de blastocisto. Aquí divulgamos que una simple rotación del embrión y de la inyección a través de la masa celular de Trans-interno (TICM) aumentó el porcentaje de ratones quiméricos del 31% al 50%, con ningún equipo adicional o formación más especializada. endogámicas de 26 diferentes clones y clones total 35 fueron inyectados en un período de 9 meses. No hubo diferencias significativas en la tasa de embarazo o índice de recuperación de embriones entre técnicas de inyección tradicional y TICM. Por lo tanto, sin ninguna alteración importante en el proceso de inyección y un simple posicionamiento del blastocisto e inyectar a través del ICM, liberación de las células madre embrionarias en la cavidad del blastocoel puede mejorar potencialmente la cantidad de producción quimérica y posterior transmisión de la línea germinal.

Tratamiento de la línea celular como el efecto aleatorio, un modelo lineal de efectos mixtos se utilizó para analizar los datos en el software (e.g. SAS (versión 9.2)). Cada análisis para el número de embriones transferidos y la línea celular. El segundo análisis también controlado por el número de cachorros que sobrevivieron.
Para este estudio, cada línea ES celular y el clon fue inyectado con tradicionales y técnica...

Watch this Scientific Journal Video about Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates at JoVE.com

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

Aquí presentamos un protocolo para aumentar la producción de quimeras sin el uso de nuevos equipos. Un cambio de orientación simple del embrión para la inyección puede aumentar el número de embriones producidos y potencialmente reducir la línea de tiempo a la transmisión de la línea germinal.

Trans-interno celular masiva inyección de células madre embrionarias conduce a mayores tasas de quimerismo

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

trans-inner-cell-mass-injection-embryonic-stem-cells-leads-to-higher

Research

JoVE Journal

Biology

10.2K vistas.  NIEHS/NIH. Aquí presentamos un protocolo para aumentar la producción de quimeras sin el uso de nuevos equipos. Un cambio de orientación simple del embrión para la inyección puede aumentar el número de embriones producidos y potencialmente reducir la línea de tiempo a la transmisión de la línea germinal.

Video: Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

Los ratones con Transnuclear predefinidos Especificidades receptor de células T contra la Toxoplasma gondii Obtenidos a través de la SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

Investigación

Inmunología e Infección

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

Los métodos para la transferencia Precisamente localizada de células o ADN en Early postimplantación embriones de ratón

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

Biología del Desarrollo

Combinational Treatment of Trichostatin A and Vitamin C Improves the Efficiency of Cloning Mice by Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer (SCNT) provides a unique opportunity to directly produce a cloned animal from a donor cell, and it requires the use of skillful techniques. Additionally, the efficiencies of cloning have remained low since the successful production of cloned animals, especially mice. There have been many attempts to improve the cloning efficiency, and trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to enhance the efficiency of cloning. Here, we report a dramatically improved cloning method in mice. This somatic cell nuclear transfer method involves usage of Hemagglutinating virus of Japan Envelope (HVJ-E), which enables easy manipulation. Moreover, the treatment using two small molecules, TSA and vitamin C (VC), with deionized bovine serum albumin (dBSA), is highly effective for embryonic development. This approach requires neither additional injection nor genetic manipulation, and thus presents a simple, suitable method for practical use. This method could become a technically feasible approach for researchers to produce genetically modified animals from cultured cells. Furthermore, it might be a useful way for the rescue of endangered animals via cloning.

We describe a dramatically improved method for mouse cloning using trichostatin A, vitamin C, and deionized bovine serum albumin. We show a simplified, reproducible protocol that supports efficient development of cloned embryos. Hence, this method could become a standardized procedure for mouse cloning.

Tratamiento combinacional de tricostatina A y la vitamina C mejora la eficiencia de la clonación de ratones por transferencia Nuclear de células somáticas

Somatic cell nuclear transfer (SCNT) provides a unique opportunity to directly produce a cloned animal from a donor cell, and it requires the use of ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

Tejido diana quimeras embrionarias: pez cebra Gástrula Trasplante de células

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Biología

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

We describe a protocol for performing an in utero transplantation (IUT) through intravenous and intra-amniotic routes of injection in the murine model. This protocol can be used to introduce cells, viral vectors, and other substances into the unique immune-tolerant fetal environment.

Intravenosa y Intra-amniótico en el útero el trasplante en el modelo murino

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

Sistema basado en la microinyección en Vivo implantación de cardiomiocitos embrionarios en el embrión aviar

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of the vertebrate genome functions. Manipulation of medaka embryos, such as dechorionation, mounting embryos for imaging and cell transplantation, are key procedures to work on both medaka and zebrafish in a laboratory. Cell transplantation examines cell autonomy of medaka mutations. Chimeras are generated by transplanting labeled cells from donor embryos into unlabeled recipient embryos. Donor cells can be transplanted to specific areas of the recipient embryos based on the fate maps 3 so that clones from transplanted cells can be integrated in the tissue of interest during development. Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. In this video, we show detailed procedures to manipulate medaka embryos.

Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. This video shows step-by-step procedures for how to manipulate medaka embryos, including dechorionation, mounting in agarose for imaging and cell transplantation for the production of chimeras. These procedures are essential to use medaka and zebrafish in a laboratory to take full advantage of their complementary features for the genetic dissection of vertebrate genome functions.

Dechorionation de embriones de medaka y trasplante de células para la generación de quimeras

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

Xenotrasplante de células madre humanas en el embrión de pollo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

Transfección de células individuales en embriones de pollo

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the efficiency in developing gene-knockout mice by inducing small indel mutation would be sufficient enough, the efficiency of embryo genome editing for making large-size DNA knock-in (KI) is still low. Therefore, in contrast to the direct KI method in embryos, gene targeting using embryonic stem cells (ESCs) followed by embryo injection to develop chimera mice still has several advantages (e.g., high throughput targeting in vitro, multi-allele manipulation, and Cre and flox gene manipulation can be carried out in a short period). In addition, strains with difficult-to-handle embryos in vitro, such as BALB/c, can also be used for ESC targeting. This protocol describes the optimized method for large-size DNA (several kb) KI in ESCs by applying CRISPR/Cas9-mediated genome editing followed by chimera mice production to develop gene-manipulated mouse models.

Here we present a protocol for developing genetically modified mouse models using embryonic stem cells, especially for large DNA knock-in (KI). This protocol is tuned up using CRISPR/Cas9 genome editing, resulting in significantly improved KI efficiency compared with the conventional homologous recombination-mediated linearized DNA targeting method.

Focalización génica altamente eficiente mediada por CRISPR/Cas9 en células madre embrionarias para desarrollar modelos de ratón manipulados genéticamente

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the ...