This work was supported by a Professional Development Grant to C.Z.P. from the Shepherd University Foundation. The authors wish to thank Brett Zirkle and Malia Deshotel for helpful discussions on the protocols, and Dr. Carol Hurney for generous assistance.

Todos los procedimientos experimentales fueron aprobados por la Universidad de Virginia Institucional Cuidado de Animales y el empleo Comisión. Nota: La figura 1 muestra una vista general esquemática de los procedimientos experimentales. 1. Preparación de ovocitos <ol><li> X. Pre-prime laevis hembras con 150 U de Mare embarazada suero de gonadotropina (PMSG) aproximadamente una semana antes de que el aislamiento de los ovocitos. Inyectar 1 ml 150 U / ml PMSG en dorsal linfático salida con 1 cc jeringa estéril con 29 G aguja. </li><li> Preparar soluciones para inyección de ovocitos y ensayo casquillo ovocito-animal. <ol><li> Preparar Ca ++ / Mg ++ exento de OR2 (OR2-): NaCl 82 mM, KCl 2,5 mM, 1,5 mM Na 2 HPO 4, 50 mM HEPES pH 7,2. </li><li> Preparar OR2: OR2- más 1 mM CaCl2 y MgCl2 1 mM. </li><li> Preparar OCM: 60% Leibovitz L15, 0,4 mg / ml BSA, 100 mg / ml de gentamicina, pH 7,8. </li><li> Prepare MBS 1x: NaCl 88 mM, 1 mM de KCl, 0,7 mM CaCl 2, 1 mM MgSO 4, 5 mM HEPES (pH 7,8), 2,5 mM NaHCO 3. </li><li> Preparar 3% de Ficoll en 1X MBS. </li><li> Preparar NAM 1x: NaCl 110 mM, KCl 2 mM, 1 mM Ca (NO 3) 2, 1 mM MgSO 4, EDTA 0,1 mM, 1 mM NaHCO 3, 2 mM Na 3 PO 4, pH 7,4. </li></ol></li><li> Anestesiar la hembra en una solución al 0,03% de acetato de sal metanosulfonato 3-aminobenzoato (MS222) en 0,1x MBS (primero disolver 0,3 g MS222 en etanol 10 ml de 95%, luego se diluye en 1 litro MBS 0,1x) mediante la colocación de rana en solución para 10-15 minutos o hasta que no responde. <ol><li> Compruebe que la anestesia es completa girando rana anestesiado sobre su espalda para asegurarse de que no responde (incompletamente ranas anestesiados mover las extremidades o girar el cuerpo más). </li></ol></li><li> Quirúrgicamente aislar fragmentos de ovario haciendo una incisión abdominal a través de la piel y la pared del cuerpo con hoja de bisturí, aislando tejido ovárico con pinzasy tijeras. Coloque fragmentos de ovario en OR2-. Cierre la pared del cuerpo con una sutura de seda 3-0 en una aguja curva de 24 mm y cierre la piel por separado con los mismos puntos de sutura. Permitir la recuperación de la hembra en 1 g / l de sal de acuario en el agua. </li><li> Con unas pinzas finas, tejido ovárico lágrima en pequeños (10 - 20 ovocitos) piezas y traslado al OR2- fresco. </li><li> Fragmentos de ovario Defolliculate en 2,0 mg / ml de colagenasa A en OR2- agitando suavemente en un agitador durante 1 hora, la transferencia a colagenasa fresco y agitar durante una hora adicional. </li><li> Lavar ovocitos 10 veces en OR2 [que contienen Ca ++ / Mg ++], descartando ovocitos pequeños. </li><li> Lave ovocitos en OCM dos veces y traslado al fresco OCM. Aislar ovocitos Etapa VI por tamaño después de una inspección visual y desechar ovocitos inmaduros (más pequeñas): Etapa VI ovocitos son más grandes que los ovocitos inmaduros con pigmentación incluso en el hemisferio animales y son de aproximadamente 1,2-1,4 mm de diámetro. </li><li> Mantener ovocitos a 18-20 ° C in OCM antes de la inyección. Nota: placas de Petri recubiertas de agarosa se pueden utilizar para minimizar la adherencia de los ovocitos a plato. </li></ol> 2. La inyección de transcripciones Library <ol><li> Preparar una biblioteca de cDNA direccional 15 usando ARN extraído en una etapa apropiada de desarrollo (por ejemplo, la etapa placa neural 14 10). Compra una biblioteca de ADNc o construir uno usando un kit comercial por la visión general en los cuatro pasos siguientes. <ol><li> Aislar aproximadamente 5 g de ARNm mediante el uso de un producto para enriquecer en poli (A) + RNA (Ver Tabla de Materiales) y siguiendo las instrucciones del fabricante. Nota: Las transcripciones que se utilizarán para producir la biblioteca de cDNA puede ser restringido a un tejido induciendo (tal como la placa neural, después de microdisección del tejido deseado), o pueden ser a partir de embriones enteros en una etapa particular. </li><li> Producir ADNc con un kit comercial, siguiendo las instrucciones del fabricante. </li><li> Ligar aproximadamente 20 ng de ADNc en un vector incluido con el kit comercial u otro vector adecuado. Un vector de plásmido apropiado incluye secuencias para la estabilidad del ARNm tales como 5 &#39;β-globina y 3 de poli (A) secuencias (pCS2 +, pTnT, pCS105). </li><li> Transformar ligación en células bacterianas competentes utilizando el protocolo recomendado por proveedor para la transformación de choque térmico. Nota: Alternativamente, una colección de (ORFeome) clones marco de lectura abierto está comercialmente disponible y puede ser usado para generar las transcripciones de inyección. </li></ol></li><li> Preparar 10 piscinas de plásmidos de desde 3 10 hasta abril 10 complejidad (desde abril 10 hasta 05 10 complejidad total). Esto representa 10 placas con 1.000 - 10.000 colonias cada una. <ol><li> Biblioteca placa de cultivo en 10 placas de 15 cm LB-ampicilina, crecer 12 - 18 horas a 37 ° C, y recoger colonias de cada por una suave presión con un esparcidor de vidrio en 7 ml LB. </li><li> Preparar un stock de glicerol a partir de 0,5 ml (añadir a 0,2 ml de glicerol estéril y se almacena a -20 y# 730; C), y el uso de los restantes 6,5 ml para preparar ADN con un estándar comercialmente disponible kit de ADN miniprep siguiendo las instrucciones del fabricante. </li></ol></li><li> Linealizar el ADN plásmido agrupada (1.0 - 2.0 g) con enzima de restricción apropiada digerir 16 a 37 C durante 1 - 1,5 h. Aislar el DNA linealizado con extracción con fenol / cloroformo seguido de precipitación con etanol y resuspensión en agua por las especificaciones del kit RNA polimerasa utilizada. Sintetizar ARN sentido con un kit de ARN polimerasa comercial siguiendo las instrucciones del fabricante. </li><li> Preparar agujas de microinyección (utilizando un extractor de aguja con un tubo capilar de vidrio) de aproximadamente 20 m de diámetro; medir puntas de aguja en un microscopio compuesto con un micrómetro ocular calibrado. Nota: La configuración del extractor de agujas deben determinarse empíricamente para producir una aguja con una punta fina de tal manera que cuando se rompen con unas pinzas finas produce el tamaño de la punta deseada. </li><li> Prepárese (empujar arcilla encapa uniforme sobre el fondo del plato) arcilla forrada 35 x 10 mm placas de Petri con ranuras paralelas para mantener ovocitos en su lugar durante la microinyección; producir ranuras con una sonda centro comercial o pipeta Pasteur fundido en la punta de la llama. Como una alternativa a la arcilla, preparar platos de agarosa haciendo muescas con un molde de elastómero 17. Ovocitos Transferir con una pipeta de gran calibre a un 3% de Ficoll en 1X MBS (aproximadamente 2 ml) en las filas de los platos de barro forradas. </li><li> Usando microinyector, llenar la aguja con 1 ng / nl ARN y ajustar el balance para producir ligera presión positiva (para evitar la elaboración de citoplasma del ovocito). </li><li> Inyectar ovocitos con aproximadamente 20 nl ARN en la región ecuatorial. Permitir 1 h para los ovocitos inyectados a permanecer en Ficoll-MBS, a continuación, transferir suavemente para 1x MBS. Incubar durante 8-24 horas a 20 ° C antes del ensayo casquillo animal. </li></ol> 3. Cap Ensayo Animal <ol><li> Preparar 3 / 4x NAM y obtener unas pinzas finas, bucle de cabello, fragmentos cubreobjetos curvas, dis barro forradashes con hendiduras en forma de copa para mantener ovocitos. Haga fragmentos cubreobjetos curvas rompiendo aparte cubreobjetos de vidrio en fragmentos pequeños (aproximadamente 1 - 2 mm x 2-4 mm) y que pasa por la llama hasta que los bordes pulir y caerse, produciendo una pieza curva. </li><li> Fertilizar X. laevis huevos 18 a través in vitro o el apareamiento natural y la cultura a la gástrula etapas (10 - 11 horas después de la fertilización a RT) en 0,1x MBS antes de la clasificación por etapas; recoger mediados de gástrula (etapa 11 - 11,5) 10 embriones. </li><li> Embriones de transferencia a una placa de Petri, aproximadamente la mitad con 3 / 4x NAM. El uso de dos pares de pinzas finas, retire la fertilización (vitelina) membrana desde gastrulae. </li><li> Transferencia de ovocitos a 3 / 4x NAM (aproximadamente 2 ml) en platos de barro forradas e inmovilizar en impresiones individuales en la arcilla, que produce impresiones para dar cabida a los embriones individuales como ranuras fueron descritos anteriormente. </li><li> Embriones de traslado hasta los platos de barro forradas y cortar las tapas animales de gastrulae usando dos pares de pinzas finas. Tenga cuidado de aislar casquillo ectodermo animales y no tejido ecuatorial 18. </li><li> Colocar un casquillo animal en el hemisferio animales de cada ovocito con la superficie interior de la tapa de los animales en contacto con el ovocito. Mantenga recombinantes juntos mediante la aplicación de fragmento de cubreobjetos de vidrio curvada y aplicando presión hacia abajo al vidrio, aplanando el ectodermo como los contactos cubreobjetos la arcilla (CAPS animal puede permanecer abierta con la capa interior expuesta a la superficie del ovocito para 6 - 8 horas o más largo ). Nota: Como alternativa, coloque la tapa del animal en una hendidura de la arcilla con su superficie interna orientada hacia arriba y coloque un ovocito en la tapa; asegurar el recombinante con pequeñas extensiones de arcilla. </li><li> Cultura a 20 ° C hasta el control de los embriones llegan a la etapa deseada para el ensayo. </li><li> Recombinante separada mediante la eliminación de cubreobjetos / arcilla y aislar ectodermo con unas pinzas y un bucle de pelo. </li><li> Fijar fragmentos ectodérmicas durante 1 hora en MEMFA (3,8%formaldehído en MEM [0.1 M MOPS pH 7.4, 2 mM EGTA, 1 mM MgSO y 4]). </li><li> Fragmentos de transferencia (y embriones de control) de MEMFA en etanol y se almacena a -20 ° C. </li></ol> 4. Análisis de la Respuesta en ectodermo por hibridación in situ (ISH) <ol><li> Preparar soluciones para ISH. <ol><li> Preparar 1x PBS: 0,01 M tampón fosfato salino, NaCl 0,138 M, pH 7,4 </li><li> Preparar PBS-Tween (PTW): 1x PBS, 0,1% de Tween-20 </li><li> Preparar la solución de 100x Denhart: 2% de BSA, 2% de polivinilpirrolidona, 2% de Ficoll </li><li> Preparar Tampón de Hibridación: 50% formamida, 5x SSC, 1 mg / ml de ARN de levadura torula, 1 mg / ml de heparina, solución de Denhart 1x, 0,1% de Tween-20, 0,1% de CHAPS, EDTA 10 mM, DEPC-H 2 O </li><li> Preparar maleico Acid Buffer (MAB): ácido maleico 100 mM, NaCl 150 mM, pH 7,5 </li><li> Preparar MAB + bloque: MAB, 2% reactivo de bloqueo (calor a 60 ° C para disolver) </li><li> Preparar fosfatasa alcalina (AP) Tampón: Tris 100 mMpH 9,5, 50 mM de MgCl 2, NaCl 100 mM, 0,1% de Tween, dH 2 O </li></ol></li><li> Prepare la sonda de ARN. <ol><li> El uso de un kit RNA polimerasa comercial y DIG-NTP mezcla, añadir a un tubo de 1,5 ml (50 l de reacción): 25,5 l DEPC-H 2 O, 10 l 5X Transcription Buffer, 2,5 l de mezcla 10x dig-NTP, 5 l 100 mM TDT, 2 l ARNsin, 2 l plantilla de ADN linealizado (~ 1 g / l), 3 l ARN polimerasa e incubar 37 ° C durante 90 minutos. </li><li> Añadir 2 l ARN polimerasa e incubar 37 ° C durante 60 minutos. </li><li> Compruebe 2 l de reacción en un 1% en gel de agarosa. </li><li> Añadir 1 l RQ1 RNasa libre de DNasa e incubar 37 ° C durante 20 minutos. </li><li> Precipitar la sonda mediante la adición de 50 l DEPC-H 2 O, 25 l 10 M acetato de amonio y 313 l de etanol. Almacenar a -20 C durante la noche (O / N) a continuación, recuperar el ARN por centrifugación a 13.800 xg durante 20 min. Lavar con 500 l 75% de etanol, girar brevemente, retireetanol y permitir pellet se seque al aire. Añadir 50 l DEPC-H 2 O. </li><li> Añadir Tampón de Hibridación a una concentración final de ~ 0,5 g / l. </li></ol></li><li> Preparar de tejidos para la hibridación. A menos que se indique lo contrario, llenar viales a la cima con cada cambio de solución descritos (aproximadamente 4 ml). <ol><li> Retire etanol a partir de viales y embriones de transferencia en 75% de etanol / PTW, entonces 50% de etanol / PTW durante 10 minutos cada uno, en posición horizontal sobre rockero. </li><li> Lavar tres veces en PTW durante 5 minutos cada uno en eje de balancín. </li><li> Transferencia a 10 mg / l tratamiento de proteinasa K en PTW; tubos de roca vertical 15 min. </li><li> Enjuague dos veces 10 minutos cada uno en 0,1 M trietanolamina pH 7.8 - tubos de roca vertical. </li><li> Añadir 12,5 l anhídrido acético para tubos y el rock verticalmente 5 min. Repita el procedimiento con anhídrido acético 12,5 l adicional durante 5 min. </li><li> Lave en PTW 5 min verticalmente en eje de balancín. </li><li> REFIX en 4% de paraformaldehído 20 min en eje de balancín. Heat Tampón de Hibridacióna 60 ° C. </li><li> Lavar tres veces en PTW durante 5 minutos cada uno en eje de balancín. </li><li> Retire todos pero ~ 1 ml de PTW de cada tubo y añadir 250 l Hyb Buffer; agitar suavemente los tubos para mezclar. Tubos de roca vertical 5 min. </li><li> Reemplazar con 60 ˚C Hyb Buffer (0,5 ml) y agitar suavemente a 60 ° C 10 min. Reemplazar con frescos Hyb Buffer y agitar a 60 ° C de dos a 4 horas. </li><li> Sonda de calor (1 ml a 0,5 mg / l en Hyb Buffer) a 60 ° C (3 min). Retire Hyb Buffer y añadir sonda para tubos. Agitar suavemente O / N a 60 ° C. </li></ol></li><li> Preparar tejidos para anticuerpos. <ol><li> Cálido Hyb Buffer y 2x SSC + 0,1% de Tween-20 soluciones a 60 ° C. </li><li> Reemplace solución sonda con Hyb Buffer (salvo sondas a -20 ° C durante 2 - 3 veces reutilización). Lavar a 60 ° C durante 10 minutos. </li><li> Lavar tres veces a 60ºC en 2x SSC-Tween (20 minutos cada uno con agitación). </li><li> Lavar tres veces a 60 ° C en 0,2x SSC-Tween (20 minutos cada uno con agitación). </li><li> Lavar dos veces en MAB (RT) durante 15 min cada uno horizontalmente en eje de balancín. </li><li> Añadir 1 ml MAB + bloque. Lávese las 2 horas verticalmente en eje de balancín. </li><li> Traslado a 1 ml MAB + bloque que contiene un anti-digoxigenina-AP 1/2000. Roca verticalmente en 4 CO / N. </li></ol></li><li> Preparar Tejidos para Color reactivo. <ol><li> Reemplace la solución de anticuerpo con MAB; lavar tres veces en el MAB 5 minutos cada uno horizontalmente en eje de balancín. </li><li> Lavar tres veces en el MAB de 1 hora cada uno horizontal en eje de balancín. </li><li> Lavar dos veces en AP Buffer 10 minutos cada uno en posición horizontal sobre rockero. </li><li> Traslado tejido para pozos múltiples bandeja de plástico, retire AP Buffer y añadir BM reactivo Púrpura (~ 1 ml). Permita reacción tenga lugar en la oscuridad 5 min de O / N. </li><li> Cuando se logra tinción apropiada, traslado de tejidos a viales de PTW. Lavar dos veces 10 minutos en cada una PTW en eje de balancín. </li><li> Fijar en solución de Bouin en 4 CO / N en eje de balancín. </li><li> Lave Bouin con tres 70% de etanol / 30% PTW lavados a TA. Proceda to captura de imagen usando epi-iluminación y una cámara montada en el microscopio, el blanqueado, si es necesario 18. </li></ol></li></ol> 5. Selección Sib y clonación <ol><li> Seleccione la piscina con la mayor actividad (mayor respuesta en el tejido ectodérmico). </li><li> Valorar (diluir con LB a la densidad apropiada) el stock de glicerol de la piscina con la mayor actividad y la placa a cabo diez nuevas placas con aproximadamente una décima parte de las colonias de la etapa previa (usando la sección 2 en el protocolo anterior). </li><li> Reducir el tamaño de la piscina hasta que la actividad se remonta a una sola colonia. </li><li> Obtener secuencia de ADN usando cebadores estándar en el vector. </li></ol>

<ol>
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(39), e1932 (2010).</li><li>Sive, H., Grainger, R., Harland, 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> Early development of.Xenopus laevis.: A laboratory manual. , (2000).</li><li>Zygar, C., Cook, T., Grainger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+activation+during+early+stages+of+lens+induction+in+Xenopus.">Gene activation during early stages of lens induction in Xenopus.</a> Development. 125, 3509-3519 (1998).</li><li>Medina-Martinez, O., Jamrich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foxe+view+of+lens+development+and+disease.">Foxe view of lens development and disease.</a> Development. 134, 1455-1463 (2007).</li><li>Amin, N., Tandon, P., Osborne, N. E., Conlon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-seq+in+the+tetraploid+Xenopus.laevis.+enables+genome-wide+insight+in+a+classic+developmental+biology+model+organism.">RNA-seq in the tetraploid Xenopus.laevis. enables genome-wide insight in a classic developmental biology model organism.</a> Methods. 66, 398-409 (2014).</li><li>Gilchrist, M., Zorn, A., Voigt, J., Smith, J., Papalopulu, N., Amaya, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+a+large+set+of+full-length+clones+from+a+Xenopus+tropicalis+EST+project.">Defining a large set of full-length clones from a Xenopus tropicalis EST project.</a> Developmental Biology. 271, 498-516 (2004).</li><li>Karpinka, 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=Xenbase,+the+Xenopus.+model+organism+database;+new+virtualized+system,+data+types+and+genomes.">Xenbase, the Xenopus. model organism database; new virtualized system, data types and genomes.</a> Nucleic Acids Research. 43, D756-D763 (2015).</li><li>Zhang, S., Li, J., Lea, R., Amaya, E., Dorey, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Functional+Genome-Wide+In+Vivo+Screen+Identifies+New+Regulators+of+Signaling+Pathways+during+Early+Xenopus+Embryogenesis.">A Functional Genome-Wide In Vivo Screen Identifies New Regulators of Signaling Pathways during Early Xenopus Embryogenesis.</a> PLoS ONE. , e79469 (2013).</li></ol>

The authors declare that they have no competing financial interests.

El método descrito aquí para la clonación funcional de genes capaces de inducir una respuesta en ectodermo competente puede ser usado para identificar una amplia gama de productos génicos. Este método se expande al trabajo anterior mediante la combinación de los ensayos de inducción de tejido con técnicas de clonación de expresión. Utilizamos las vías metabólicas de Xenopus el ovocito como una fuente de producción de factores de inducción de, directa o indirectamente, después de la inyección de ...

Forward genetic approaches to identify genes of interest through their function or loss-of-function1,2 are an integral part of understanding complex patterning events in development. Coupled with powerful reverse genetic techniques available to an ever-widening array of systems and researchers3-5, it is now possible to identify genes with a key functional role in a pathway and then elucidate that function at the cellular level and in interaction with other gene products. One approach to functionally identifying genes of interest that has yielded many key findings in the past is expression cloning6,7.
Our recent aim8 was to identify early lens-inductive factors, since it has been demonstrated that initial steps in the vertebrate lens-inductive process occur as early as gastrula stages. To that end, we used the transiently lens-competent9 animal cap ectoderm (stage 11-11.510) of Xenopus embryos as responding tissue for induction, and the stage VI Xenopus oocyte as a source of production for the inducing factors.
The following protocol builds on the expression cloning and sib selection protocols of Smith and Harland6,7, also successfully used by others11-13. In our oocyte expression system (first utilized for production of inducing factors by Lustig and Kirschner14), pools of injected transcripts capable of directly or indirectly causing the oocytes to produce factors that elicit a lens-inductive response in animal cap ectoderm are selected for and identified. Since the system is useful for expressing secreted inducing molecules directly (oocyte-injected INHBB mRNA causes mesoderm induction in mesoderm-competent animal cap ectoderm8), we originally expected the screening procedure to be useful chiefly for identification of paracrine factors. However, since we identified a nuclear factor in our screen (ldb18), it is clear that the system can be used to identify a wide variety of molecules such as transcriptional or translational regulatory factors, miRNAs, cofactors, or juxtacrine factors.

<table><tbody><tr><td>12/101 Antibody</td><td>Developmental Studies Hybridoma Bank</td><td>12/101</td><td>Monoclonal antibody for detection of muscle tissue</td></tr><tr><td>20x SSC Buffer</td><td>Sigma</td><td>S6639</td><td>for ISH</td></tr><tr><td>Acetic anhydride</td><td>Sigma</td><td>A6404</td><td>for ISH</td></tr><tr><td>Anti-Dig-AP</td><td>Roche</td><td>11093274910</td><td>for ISH</td></tr><tr><td>Aurum Plasmid Mini Kit</td><td>Bio-Rad</td><td>732-6400</td><td>Plasmid DNA purification</td></tr><tr><td>Blocking Reagent</td><td>Roche</td><td>11096176001</td><td>for ISH</td></tr><tr><td>BM Purple</td><td>Roche</td><td>11442074001</td><td>for ISH</td></tr><tr><td>Boekel Hybridization Oven</td><td>Fisher Scientific</td><td>13-245-121</td><td>for ISH</td></tr><tr><td>Bouin&#039;s Solution</td><td>Sigma</td><td>HT10132</td><td>for ISH</td></tr><tr><td>BSA</td><td>Sigma</td><td>A9647</td><td>for OCM</td></tr><tr><td>CHAPS</td><td>Sigma</td><td>C3023</td><td>for ISH</td></tr><tr><td>Collagenase A</td><td>Roche</td><td>10103578001</td><td>Defolliculation of oocytes</td></tr><tr><td>Cysteine</td><td>Sigma</td><td>C121800</td><td>Dejelly embryos</td></tr><tr><td>DEPC-H2O</td><td>Fisher Scientific</td><td>BP5611</td><td>for ISH</td></tr><tr><td>Dig-RNA Labeling Mix</td><td>Roche</td><td>11277073910</td><td>for ISH probes</td></tr><tr><td>Dumont #5 forceps</td><td>World Precision Instruments</td><td>500233</td><td>for Vitelline envelope removal</td></tr><tr><td>Ethyl 3-aminobenzoate</td><td>Sigma</td><td>A5040</td><td>MS222 anesthetic</td></tr><tr><td>Ficoll PM 400</td><td>Sigma</td><td>F4375</td><td>for Injection media</td></tr><tr><td>Formamide</td><td>Sigma</td><td>F9037</td><td>for ISH</td></tr><tr><td>Gentamicin sulfate</td><td>Sigma</td><td>G1914</td><td>for OCM</td></tr><tr><td>Glass capillaries</td><td>World Precision Instruments</td><td>4878</td><td>3.5&quot; long, I,D, 0.530 mm</td></tr><tr><td>Glass sample vials</td><td>Fisher Scientific</td><td>06-408B</td><td>for ISH</td></tr><tr><td>Hair loop</td><td></td><td></td><td>Hair affixed in pasteur pipette for tissue manipulation</td></tr><tr><td>Heparin sodium salt</td><td>Sigma</td><td>H4784</td><td>for ISH</td></tr><tr><td>Injector Nanoliter 2010</td><td>World Precision Instruments</td><td>Nanoliter 2010</td><td>Microprocessor-controlled microinjector</td></tr><tr><td>Instant Ocean</td><td>Carolina</td><td>972433</td><td>Aquarium Salt for frog recovery</td></tr><tr><td>IRBG XGC Xenopus verified full-length cam cDNA</td><td>Source Bioscience</td><td>989_IRBG</td><td>cDNA library&amp;nbsp;</td></tr><tr><td>LB Agar plates with 100 &amp;micro;g/ml Ampicillin</td><td>Teknova</td><td>L5004</td><td>150 mm pre-poured LB-Amp plates for sib selection</td></tr><tr><td>LB Luria Broth</td><td>Teknova</td><td>L8650</td><td>LB for collecting colonies in sib selection from plates and dilution of cultures</td></tr><tr><td>Magnetic mRNA Isolation Kit</td><td>New England BioLabs</td><td>S1550S</td><td>for isolation of poly(A)-enriched RNA</td></tr><tr><td>Maleic Acid</td><td>Sigma</td><td>M0375</td><td>for ISH</td></tr><tr><td>Manual Microfil Micromanipulator</td><td>World Precision Instruments</td><td>M3310R</td><td>Manual micromanipulator</td></tr><tr><td>Nutating Mixer</td><td>Fisher Scientific</td><td>22-363-152</td><td>Rocker for ISH</td></tr><tr><td>Permoplast</td><td>Nasco</td><td>SB33495M</td><td>Clay for injection and dissection dishes</td></tr><tr><td>Phosphate Buffered Saline</td><td>Sigma</td><td>P5368</td><td>for ISH</td></tr><tr><td>PMSG</td><td>Sigma</td><td>G4877</td><td>to stimulate oocyte development</td></tr><tr><td>Polyvinylpyrrolidone</td><td>Sigma</td><td>PVP40</td><td>for ISH</td></tr><tr><td>Programmable Puller</td><td>World Precision Instruments</td><td>PUL-1000</td><td>Micropipette needle puller</td></tr><tr><td>Proteinase K</td><td>Sigma</td><td>P6556</td><td>for ISH</td></tr><tr><td>pTnT Vector</td><td>Promega</td><td>L5610</td><td>cDNA library construction</td></tr><tr><td>Riboprobe Combination System</td><td>Promega</td><td>P1450</td><td>in vitro transcription</td></tr><tr><td>Superscript Full Length cDNA Library Construction Kit</td><td>Life Technologies</td><td>18248013</td><td>kit for cDNA library construction</td></tr><tr><td>Sutures, 3-0 silk</td><td>Fisher Scientific</td><td>19-037-516</td><td>Suture thread and needle for post-oocyte removal</td></tr><tr><td>Torula RNA</td><td>Sigma</td><td>R3629</td><td>for ISH</td></tr><tr><td>Triethanolamine</td><td>Sigma</td><td>T1502</td><td>for ISH</td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P9416</td><td>for ISH</td></tr><tr><td>Universal RiboClone cDNA Synthesis System</td><td>Promega</td><td>C4360</td><td>alternative kit for cDNA library construction</td></tr><tr><td>Xenopus Full ORF Entry Clones - ORFeome Collaboration</td><td>Source Bioscience</td><td>5055_XenORFeome</td><td>ORFeome Clones</td></tr><tr><td>XL2-Blue Ultracompetent Cells</td><td>Agilent Technologies</td><td>200150</td><td>cells for transformation of cDNA library</td></tr></tbody></table>

functional cloning using a xenopus oocyte expression system

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

En respuesta a la expresión del ARNm inyectado en oocitos, respondiendo tejido casquillo animal se sometió a ensayo para la expresión de Otx2 mediante hibridación in situ (Figura 2 y Tabla 1); Otx2 se expresa en el ectodermo lente presuntiva (PLE) a partir de cierre del tubo neural a través de placoda del cristalino engrosamiento 19. Sin embargo, desde Otx2 se expresa también en el ectodermo neural anterior, así como ...

Watch this Scientific Journal Video about Functional Cloning Using a Xenopus Oocyte Expression System at JoVE.com

Functional Cloning Using a Xenopus Oocyte Expression System

Clonación funcional utilizando una<em&gt; Xenopus</em&gt; Sistema de Expresión de ovocitos

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

functional-cloning-using-a-xenopus-oocyte-expression-system

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

Functional Cloning Using a Xenopus Oocyte Expression System

8.0K vistas.  Shepherd University. We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

Video: Functional Cloning Using a Xenopus Oocyte Expression System

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

La manipulación y<em&gt; In Vitro</em&gt; La maduración de<em&gt; Xenopus laevis</em&gt; Los ovocitos, seguido de inyección intracitoplasmática de espermatozoides, para estudiar el desarrollo embrionario

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Investigación

Biología del Desarrollo

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

Uso del análisis confocal de<em&gt; Xenopus laevis</em&gt; Encargado de investigar moduladores de Wnt y Shh morfógeno Degradados

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the African clawed frog, Xenopus, a longstanding model organism for biomedical research. Traditional breeding schemes for creating homozygous mutant lines with CRISPR/Cas9-targeted mutagenesis have several time-consuming and laborious steps. To facilitate the creation of mutant embryos, particularly to overcome the obstacles associated with knocking out genes that are essential for embryogenesis, a new method called leapfrogging was developed. This technique leverages the robustness of Xenopus embryos to &#34;cut and paste&#34; embryological methods. Leapfrogging utilizes the transfer of primordial germ cells (PGCs) from efficiently-mutagenized donor embryos into PGC-ablated wildtype siblings. This method allows for the efficient mutation of essential genes by creating chimeric animals with wildtype somatic cells that carry a mutant germline. When two F0 animals carrying &#34;leapfrog transplants&#34; (i.e., mutant germ cells) are intercrossed, they produce homozygous, or compound heterozygous, null F1 embryos, thus saving a full generation time to obtain phenotypic data. Leapfrogging also provides a new approach for analyzing maternal effect genes, which are refractory to F0 phenotypic analysis following CRISPR/Cas9 mutagenesis. This manuscript details the method of leapfrogging, with special emphasis on how to successfully perform PGC transplantation.

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

Genes essential for survival pose technical hurdles for creating mutant lines. Leapfrogging circumvents lethality by combining genome editing with primordial germ cell transplantation to create wild-type animals carrying germline mutations. Leapfrogging also permits the efficient generation of homozygous null mutants in the F1 generation. Here, the transplantation step is demonstrated.

Trasplante de células de germen primordiales para basada en CRISPR/Cas9 Leapfrogging en Xenopus

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the ...

Genética

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

Celular-como Stem<em&gt; Xenopus</em&gt; Embrionarias explantes para Estudiar Características Temprana del Desarrollo Neural<em&gt; In Vitro</em&gt; Y<em&gt; En Vivo</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Microinyección de oocitos de Xenopus laevis

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Biología

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

Microinyección de ADN en los oculares en embriones de Xenopus laevis e imágenes de gFP que expresan los árboles axonales ópticos en los renacuajos de Xenopus vivos

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

La fertilización de Oocitos de Xenopus Utilizando el método de transferencia de acogida

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Células vivas, imágenes y análisis cuantitativo de las células epiteliales embrionarias en Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

La producción de transgénicos Xenopus laevis Integración mediada por la enzima de restricción y transferencia nuclear

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an oocyte by a spermatozoon creates a totipotent zygote. Recently, several publications have reported that haploid embryonic stem cells (haESCs) can be a substitute for gametic genomes and contribute to embryos, which develop into mice. Here, we present a protocol to apply parthenogenetic haESCs as a substitute of sperm to construct embryos by intracytoplasmic injection into oocytes. This protocol consists of steps for preparing haESCs as sperm replacement, for injection of haESC chromosomes into oocytes, and for culture of semi-cloned embryos. The embryos can yield fertile semi-cloned mice after embryo transfer. Using haESCs as sperm replacement facilitates genome editing in the germline, studies of embryonic development, and investigation of genomic imprinting.

This article aims to demonstrate the use of parthenogenetic haploid embryonic stem cells as a substitute for sperm for the construction of semi-cloned embryos.

Aplicación de células madre embrionarias haploides partegenéticas del ratón como sustituto de los espermatozoides

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an ...

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