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 os procedimentos experimentais foram aprovados pela Universidade de Virginia Institutional Animal Care e do Comitê Use. Nota: A Figura 1 mostra uma vista geral esquemática dos procedimentos experimentais. 1. Preparação de oócitos <ol><li> X. Pré-prime laevis fêmeas com 150 U de Pregnant Mare Serum gonadotrofina (PMSG) cerca de uma semana antes do isolamento do oócito. Injectar 1 ml de 150 U / ml PMSG em dorsal saco linfático com 1 cc seringa estéril com 29 g de agulhas. </li><li> Preparar soluções para injecção de oócitos e oócito animal ensaio cap. <ol><li> Prepare de Ca ++ / Mg ++ livre de OR2 (OR2-): NaCl 82 mM, KCl 2,5 mM, 1,5 mM de Na 2 HPO 4, 50 mM de HEPES pH 7,2. </li><li> Prepare OR2: OR2- adicionalmente 1 mM de CaCl2 e 1 mM de MgCl2. </li><li> Prepare OCM: 60% Leibovitz L15, 0,4 mg / mL de BSA, 100? G / ml de gentamicina, pH 7,8. </li><li> Prépare MBS 1x: NaCl 88 mM, KCl 1 mM, 0,7 mM de CaCl2, 1 mM de MgSO4, 5 mM de HEPES (pH 7,8), 2,5 mM de NaHCO3. </li><li> Prepare a 3% de Ficoll em 1x MBS. </li><li> Prepare NAM 1x: NaCl 110 mM, KCl 2 mM, 1 mM de Ca (NO3) 2, 1 mM de MgSO4, 0,1 mM de EDTA, 1 mM de NaHCO 3, 2 mM Na 3 PO 4, pH 7,4. </li></ol></li><li> Anestesiar a fêmea em uma solução de 0,03% de éster etílico do sal metanossulfonato de 3-aminobenzoato de metilo (MS222) em MBS 0,1x (dissolver primeiro 0,3 g MS222 em 10 ml de etanol a 95%, depois dilui-se em 1 L de MBS 0,1x) por colocação em solução de rã 10 - 15 minutos ou até que não responde. <ol><li> Verifique que a anestesia é completa, rodando sapo anestesiado sobre suas costas para garantir que ele não responde (incompletamente rãs anestesiados mover os membros ou virar corpo ao longo). </li></ol></li><li> Cirurgicamente isolar fragmentos de ovário, fazendo uma incisão abdominal através da pele e da parede do corpo com lâmina de bisturi, isolando tecido ovariano com uma pinçae tesouras. Coloque fragmentos ovarianos em OR2-. Fechar a parede do corpo com uma sutura de seda 3-0 em uma agulha curva 24 mm e feche a pele separadamente com os mesmos fios. Permitir a recuperação da fêmea no 1 g / L de sal na água do aquário. </li><li> Usando uma pinça fina, tecido ovariano rasgo em pequenas (10 - 20 oócitos) peças e transferência para OR2- fresco. </li><li> Fragmentos ovarianos Defolliculate em 2,0 mg / ml de colagenase A em OR2- agitando suavemente num agitador durante 1 hora, a transferência de colagenase fresco e agitação durante uma hora adicional. </li><li> Wash ovócitos 10 vezes em OR2 [contendo Ca ++ / Mg ++], descartando oócitos menores. </li><li> Lave oócitos em OCM duas vezes e transferir a nova OCM. Isolar oócitos Fase VI por tamanho após a inspeção visual e descartar oócitos imaturos (menores): ovócitos Fase VI são maiores do que os oócitos imaturos, mesmo com pigmentação no hemisfério animais e são cerca de 1,2-1,4 mm de diâmetro. </li><li> Manter oócitos a 18 - 20 ° C iN OCM antes da injecção. Nota: As placas de Petri revestidas com agarose pode ser utilizado para minimizar a aderência de oócitos de prato. </li></ol> 2. Injecção da Biblioteca Transcrições <ol><li> Prepara-se uma biblioteca de cDNA direccional 15 utilizando ARN extraído numa fase adequada do desenvolvimento (por exemplo, placa neural fase 14 10). Comprar uma biblioteca de cDNA ou construir um usando um kit comercial acordo com a visão geral nos quatro passos abaixo. <ol><li> Isolar cerca de 5 ug de ARNm usando um produto para enriquecer para poli (A) + ARN (Ver tabela de materiais) e seguindo as instruções do fabricante. Nota: Os transcritos para ser usado para produzir a biblioteca de ADNc pode ser restrita a um tecido indução (tais como a placa neural, seguindo microdissecação do tecido desejado), ou pode ser a partir de embriões inteiros numa fase particular. </li><li> Produção de ADNc com um kit comercial, seguindo as instruções do fabricante. </li><li> Ligadura, aproximadamente, 20 ng de cDNA para um vector incluído com o kit comercial ou outro vector adequado. Um vector plasmídeo apropriado inclui sequências de estabilidade do ARNm, tal como 5 &#39;β-globina e 3 poli (A) (sequências pCS2 +, PTNT, pCS105). </li><li> Transforme ligação em células bacterianas competentes que utilizam o protocolo recomendado pelo fornecedor para a transformação de choque térmico. Nota: Alternativamente, um conjunto de grelha de leitura aberta (ORFeome clones) está comercialmente disponível e pode ser usado para gerar transcrições para injecção. </li></ol></li><li> Preparar 10 piscinas de plasmídeos de 10 para 3 10 4 complexidade (10 4 10 5 para a complexidade total). Isto representa 10 placas com 1.000 - 10.000 colónias cada. <ol><li> Biblioteca placa de cultura em 10 placas de 15 cm LB-ampicilina, crescer 12-18 horas a 37 C, e recolher colónias de cada por uma leve pressão com um espalhador de vidro em 7 ml LB. </li><li> Prepare um estoque de glicerol de 0,5 ml (adicionar aos 0,2 ml de glicerol e armazenar estéril a -20 &amp;# 730; C), e utilizar os restantes 6,5 ml para preparar DNA com um padrão disponível comercialmente kit DNA miniprep seguindo as instruções do fabricante. </li></ol></li><li> Linearizar DNA plasmídeo em pool (1,0-2,0 mg) com restrição apropriada enzima digerir 16 a 37 ° C durante 1-1,5 h. Isolar o ADN linearizado com extracção com fenol / clorofórmio seguido de precipitação com etanol e ressuspensão em água de acordo com as especificações do kit de ARN-polimerase utilizada. Sintetizar RNA sentido com ARN-polimerase de um kit comercial, seguindo as instruções do fabricante. </li><li> Prepare agulhas para microinjeção (usando um extrator de agulha com tubos capilares de vidro) de aproximadamente 20 mm de diâmetro; medir pontas de agulha em um microscópio composto com um micrômetro ocular calibrada. Nota: As configurações do extrator de agulhas deve ser determinada empiricamente para produzir uma agulha com uma ponta fina de tal forma que quando quebrado com uma pinça fina produz o tamanho da ponta desejado. </li><li> Prepare (empurrar barro emcamada uniforme na parte inferior do prato) 35 x 10 mm placas de Petri com sulcos paralelos para manter oócitos no lugar durante a microinjeção forrada com argila; produzir sulcos com uma sonda de shopping ou pipeta Pasteur fundido na ponta em chamas. Como uma alternativa para argila, preparar pratos agarose fazendo recortes com um molde de elastômero 17. Oócitos meio de uma pipeta de todo o furo para 3% Ficoll em 1x MBS (aproximadamente 2 ml) em filas nos pratos forrado de barro. </li><li> Usando microinjector, preencher agulha com 1 ng / nl RNA e ajustar o equilíbrio para produzir uma ligeira pressão positiva (para evitar a elaboração de citoplasma de oócitos). </li><li> Injetar oócitos com aproximadamente 20 nl RNA na região equatorial. Permitir uma hora para oócitos injectados para permanecer em Ficoll-MBS, em seguida, transferir cuidadosamente para 1x MBS. Incubar durante 8 - 24 horas a 20 ° C antes do ensaio tampão animal. </li></ol> 3. animal Cap Assay <ol><li> Prepare 3 / 4x NAM e obter uma pinça fina, laço de cabelo, fragmentos lamela curvas, dis-alinhado argilahes com recortes em forma de taça para manter oócitos. Faça fragmentos lamela curvas desmembrando-lamelas de vidro em pequenos fragmentos (aproximadamente 1 - 2 mm x 2-4 mm) e passando pela chama até que as bordas polonês e inclinar-se, produzindo uma peça curvada. </li><li> Fertilize X. laevis ovos 18 através in vitro ou do acasalamento natural e cultura para gastrula fases (10-11 horas pós-fertilização no RT) em 0,1x MBS antes da ordenação por etapa; recolher meados de gástrula (fase 11 - 11,5) 10 embriões. </li><li> Embriões de transferência para uma placa de Petri aproximadamente meio cheio com 3 / 4x NAM. Usando dois pares de uma pinça fina, retire a fertilização (vitelline) membrana de gastrulae. </li><li> Transferência de oócitos a 3 / 4x NAM (aproximadamente 2 ml) em placas forradas de argila e imobilizar em impressões individuais na argila, produzindo impressões para acomodar embriões individuais como ranhuras foram descritos acima. </li><li> Embriões de transferência para os pratos forrado de barro e cortar tampas de animais fora gastrulae usando dois pares de fórceps finos. Tome cuidado para isolar animais cap ectoderma somente e não tecido equatorial 18. </li><li> Colocar uma tampa de animal no hemisfério animal de cada oócito com a superfície interior da tampa de animais contactar o oócito. Segurar recombinantes em conjunto aplicando fragmento lamela de vidro curvo e a aplicação de pressão descendente para o vidro, achatando o ectoderme como os contactos lamela da argila (tampões animais podem permanecer aberta com a camada interior exposta à superfície do oócito para 6 - 8 h ou mais ). Nota: Como alternativa, coloque a tampa de animais em um recuo de barro com sua superfície interna voltada para cima e coloque um oócito na tampa; fixar o recombinante com pequenas extensões de argila. </li><li> Cultura a 20 ° C até a embriões de controlo atingiram a fase desejada para o ensaio. </li><li> Recombinante separada, removendo lamela / argila e isolando ectoderma com uma pinça e um laço de cabelo. </li><li> Corrigir fragmentos ectodérmicas durante 1 hora em MEMFA (3,8%formaldeído em MEM [MOPS 0,1 M pH 7,4, EGTA 2 mM, e 1 mM de MgSO 4]). </li><li> Fragmentos de transferência de embriões (e controle) de MEMFA em etanol e armazenar a -20 C. </li></ol> 4. Análise de Resposta na ectoderme por hibridação in situ (ISH) <ol><li> Preparar soluções para ISH. <ol><li> Prepare 1x PBS: 0,01 M de solução salina tamponada de fosfato, NaCl 0,138 M, pH 7,4 </li><li> Prepare PBS-Tween (PTW): 1x PBS, 0,1% de Tween-20 </li><li> Preparar a solução de Denhart 100x: 2% de BSA, 2% de polivinilpirrolidona, 2% de Ficoll </li><li> Preparar tampão de hibridização: 50% formamida, 5x SSC, 1 mg / ml de ARN de levedura torula, 1 ug / ml de heparina, solução de Denhart 1x, 0,1% de CHAPS a 0,1%, EDTA 10 mM, Tween-20,, DEPC-H2O </li><li> Preparar tampão de ácido maleico (MAB): 100 mM de ácido maleico, NaCl 150 mM, pH 7,5 </li><li> Prepare MAB + bloco: MAB, 2% reagente de bloqueio (calor a 60 ° C para dissolver) </li><li> Prepare a fosfatase alcalina (FA) Tampão: 100 mM de TrispH 9,5, 50 mM de MgCl2, 100 mM de NaCl, 0,1% de Tween, dH2O </li></ol></li><li> Prepara-se o ARN da sonda. <ol><li> Utilizando um kit de ARN polimerase comercial e cavar-NTP mistura, adicionar a um tubo de 1,5 ml (50 reacção ul): 25,5 mL de DEPC-H2O, 10 uL de 5X de Transcrição Tampão, 2,5 ul 10x DIG-mistura de NTP, 5 ul de 100 mM DTT, 2 ul de RNAsin, 2 ul de molde de ADN linearizado (~ 1 ug / ul), 3 ul de ARN-polimerase de 37 ° C e incubar durante 90 min. </li><li> Adicionar 2 l de ARN polimerase e incubar 37 ° C durante 60 min. </li><li> Verifique 2 ul de reacção sobre 1% de gel de agarose. </li><li> Adicionar 1 mL RQ1 RNase livre de DNase e incubar 37 ° C durante 20 min. </li><li> Precipitar a sonda por adição de 50 ul de DEPC-H2O, 25 mL de 10 M de acetato de amónio e 313 mL de etanol. Armazenar a -20 C durante a noite (O / N), em seguida, recuperar o ARN por centrifugação a 13.800 xg durante 20 min. Lavar com 500 ul de etanol 75%, girar rapidamente, removaetanol e permitir pellet secar ao ar. Adicionar 50 ul de DEPC-H2O </li><li> Adicionar tampão de hibridação a uma concentração final de ~ 0,5 ug / uL. </li></ol></li><li> Prepare tecido por hibridação. Salvo disposição em contrário, encher frascos ao topo com cada mudança de solução descritos (cerca de 4 ml). <ol><li> Remover o etanol e a partir de frascos de transferência de embriões em 75% de etanol / PTW, em seguida, 50% de etanol / PTW durante 10 min cada, horizontalmente no balancim. </li><li> Lavar três vezes em PTW durante 5 min cada em balancim. </li><li> Transferir para 10 ng / mL tratamento Proteinase K em PTW; tubos de rock verticalmente 15 min. </li><li> Lavar duas vezes em cada 10 min trietanolamina 0,1 M de pH 7,8 - tubos de rocha verticalmente. </li><li> Adicionar 12,5 ul anidrido acético para tubos e rock verticalmente 5 min. Repita com anidrido acético adicional de 12,5 ul por 5 min. </li><li> Lavar em PTW 5 min verticalmente no balancim. </li><li> Refix em paraformaldeído a 4% em 20 min balancim. Calor Tampão de Hibridizaçãoa 60 ° C. </li><li> Lavar três vezes em PTW durante 5 min cada em balancim. </li><li> Remova todos, mas ~ 1 ml de PTW de cada tubo e adicionar 250 ul Hyb tampão; Agite cuidadosamente tubos para misturar. Tubos de rocha vertical 5 min. </li><li> Substitua com 60 ° C Hyb Buffer (0,5 ml) e agitar suavemente a 60 ° C 10 min. Substitua com frescos Hyb tampão e agitar a 60 ° C de duas a quatro horas. </li><li> Sonda de calor (1 ml a 0,5 mg / mL em tampão de Hib) a 60 ° C (3 min). Remover Hyb tampão e adicionar sonda para tubos. Agitar suavemente O / N a 60 ° C. </li></ol></li><li> Prepare Tissue para o Anticorpo. <ol><li> Quente Hyb buffer e 2x SSC + 0,1% Tween-20 soluções a 60 C. </li><li> Substitua solução de sonda com Hyb Buffer (sondas salvar a -20 C durante 2 - 3x reutilização). Lavar a 60 ° C durante 10 min. </li><li> Lavar três vezes a 60 ° C em 2x SSC-Tween (20 min cada, com agitação). </li><li> Lavar três vezes a 60 ° C em 0,2x SSC-Tween (20 min cada, com agitação). </li><li> Lavar duas vezes em MAB (RT) durante 15 min cada horizontalmente no balancim. </li><li> Adicione 1 ml MAB + bloco. Lave 2 horas verticalmente no balancim. </li><li> Transferência para 1 ml MAB + bloco que contém um anti-digoxigenina-AP 1 / 2.000. Rocha verticalmente a 4 CO / N. </li></ol></li><li> Prepare tecido por reagente de cor. <ol><li> Substituir solução de anticorpo com MAB; lavar três vezes em 5 min cada MAB horizontalmente no balancim. </li><li> Lavar três vezes em cada MAB 1 h horizontalmente no balancim. </li><li> Lavar duas vezes em tampão de AP 10 min cada horizontalmente no balancim. </li><li> Transferir para tecido de múltiplas bem bandeja de plástico, remover e adicionar tampão de AP BM reagente roxa (~ 1 mL). Permitir que a reacção prossiga na obscuridade 5 min a O / N. </li><li> Quando a coloração adequada é alcançado, transferir tecido para frascos de PTW. Lavar duas vezes 10 minutos cada em PTW no balancim. </li><li> Corrigir em solução de Bouin a 4 CO / N no balancim. </li><li> Lave a Bouin com três de 70% de etanol / 30% PTW lavagens em temperatura ambiente. Prossiga tcaptura de imagem usando o epi-iluminação e uma câmera montada no microscópio, branqueamento, se necessário 18. </li></ol></li></ol> 5. Sib Seleção e Clonagem <ol><li> Selecionar piscina com a maior atividade (maior resposta no tecido ectodérmica). </li><li> Titular (diluir com LB a densidade adequada) o estoque de glicerol para a piscina com a maior atividade e placa out dez novas placas com aproximadamente um décimo das colônias da etapa anterior (usando a seção 2 em protocolo acima). </li><li> Reduzir o tamanho da piscina até que a atividade é atribuída a única colônia. </li><li> Obter sequência de ADN utilizando iniciadores convencionais no vector. </li></ol>

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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.

O método aqui descrito para a clonagem de genes funcional capaz de induzir uma resposta em ectoderme competente pode ser utilizado para identificar uma grande variedade de produtos de genes. Este método expande trabalho passado, combinando ensaios de indução de tecido-com técnicas de clonagem de expressão. Nós utilizamos as vias metabólicas do oócito de Xenopus como uma fonte de produção de factores de induzir, directa ou indirectamente, após a injecção de ARN. Isto, em combinação com a utiliza?...

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.

Em resposta à expressão do ARNm injectado em oócitos, respondendo tecido tampão animal foi ensaiado quanto à expressão de OTX2 por hibridação in situ (Figura 2 e Tabela 1); OTX2 é expressa em ectoderme lente presuntivo (PLE) de fecho do tubo neural através placï¿½io lente espessamento 19. No entanto, uma vez que OTX2 é também expressa em ectoderme neural anterior, bem como ectoderme cabeça não neurais fora d...

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

Clonagem Funcional Utilizando um<em&gt; Xenopus</em&gt; Oocyte Sistema de Expressão

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 visualizações.  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.

Vídeo: 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.

Manipulação e<em&gt; In Vitro</em&gt; Maturação<em&gt; Xenopus laevis</em&gt; Oócitos, seguido de injeção intracitoplasmática de espermatozóide, para estudar o desenvolvimento embrionário

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

Biologia do Desenvolvimento

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.

Utilizando Análise de confocal<em&gt; Xenopus laevis</em&gt; Para Investigar moduladores de Wnt e Shh morfogénio gradientes

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.

Transplante de células de germes primordial para baseado em CRISPR/Cas9 saltos em 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.

Stem Cell-like<em&gt; Xenopus</em&gt; Embrionárias explantes Estudar características Neural desenvolvimento precoce<em&gt; In Vitro</em&gt; E<em&gt; In 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.

Microinjeção de Oócitos Xenopus laevis

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

Biologia

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.

Microinjection do ADN em Eyebuds em embriões do laevis de Xenopus e imagem latente de GFP que expressa Arbors axonal óticos em girinos intact, vivendo de Xenopus

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.

Fertilização de Oócitos Xenopus Utilizando o método de transferência do host

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.

Em células vivas, Imagem e Análise Quantitativa de células embrionárias epiteliais em 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.

Produção de transgênicos Xenopus laevis Por Restrição Integração Mediada por Enzima e Transplante 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.

Aplicação de Células-Tronco Embrionárias Haploid Partehenogenéticas do Rato como um substituto do esperma

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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