Esta pesquisa foi apoiada [em parte] pelo programa de pesquisa Intramural do NIH, Instituto Nacional de Ciências de saúde ambiental. Agradecimento especial a Shyamal Peddada para análise estatística. Gostaríamos também de agradecer Humphrey Yao, Gary Bird e Yuki Arao para revisão deste manuscrito, e Franco DeMayo por seu contínuo apoio e conselhos.

Todas as operações foram feitas como parte da operação normal da instalação NIEHS Knock Out Mouse Core. Todos os ratos foram mantidos em um 12:12 ciclo h luz: escuro e foram alimentar uma dieta de NIH-31 e água ad libitum. Todos os experimentos foram realizados em conformidade com o Comité de utilização do nacional Instituto de Ciências de saúde ambiental e cuidado Animal.
1. preparação animal
<ol><li>Raça de ratos do doador, B6 (Cg)-Tyr < c-2J > /J ratos fêmeas em 8-10 semanas de idade naturalmente a B6 (Cg)-Tyr < c-2J > /J camundongos machos entre 8 e 26 semanas de idade. Verificar visualmente se conecta na manhã seguinte (E0.5).</li>
	<li>Raça de ratos destinatários, Swiss Webster ratos fêmeas entre 6 e 9 semanas de idade são naturalmente criados para camundongos machos de Swiss Webster vasectomizados. Verificar visualmente se conecta na manhã seguinte (E0.5).</li>
</ol>2. preparação da pilha ES
<ol><li>Para as células de AB2.2 ES 129-derieved, cultura de células em DMEM suplementado com 15% FBS, 1% glutamina, 1% Penn/estreptococos e 0,1% β-Mercaptoetanol. Cultura de todas as outras células ES segundo as instruções do fornecedor de celular ES, usando seus protocolos recomendados.</li>
</ol>3. embrião coleção
<ol><li>
Dissecação de animais
	<ol>
<li>Preparar vários pratos de 60 mm, aproximadamente 1 por 5 ratos mais 3 extra, com mídia de coleção de explosão de 10 mL (BCM) (10% FBS em DMEM), e um 4 bom prato com 0,5 mL BCM por bem. Equilibrar a 37 ° C, 5% de CO2.</li>
		<li>Eutanásia em B6 (Cg)-Tyr < c-2J > /J camundongos fêmeas no E3.5 usando asfixia de CO2 , com um caudal de 10%.</li>
		<li>Após a eutanásia, coloque os ratos na posição supina e molhar o abdômen completamente com etanol a 70%.</li>
		<li>Façam a primeira incisão através da pele somente, no ponto médio do abdômen, fazendo a abertura tão ampla quanto possível. Em seguida abra através da parede abdominal, fazendo a incisão tão ampla quanto possível. Nota: A fazer isto em dois passos ajuda a reduzir ou eliminar a contaminação da pele.</li>
		<li>Começando com um ovário e segurando do tecido do ovário com fórceps rombudo, remover cuidadosamente o ovário, oviduto e útero, tomar cuidado extra para manter o tecido adiposo a um mínimo. Remova o útero como uma única unidade, ou como chifres individuais, com nenhuma mudança no procedimento a seguir.</li>
		<li>Após a remoção, coloque o útero em um prato de 60 mm que contém a mídia de coleção de explosão.</li>
	</ol>
</li>
	<li>
Remoção do embrião
	<ol>
<li>Preparar uma seringa de 10ml cheia de BCM e se encaixam com uma agulha de 25 G.</li>
		<li>Um útero de cada vez, removê-los do prato de exploração do BCM, exterminá-los em um lenço de papel para remover o excesso de sangue e coloque em um prato de 60 mm em um pequeno aumento, microscópio de dissecção.</li>
		<li>Mantendo cuidadosamente e suavemente o útero, logo abaixo da extremidade distal (perto do oviduto) com pinça de dissecação, introduza a agulha para o centro do útero em como paralelo um avião quanto possível. Insira a agulha passando as pontas da pinça.</li>
		<li>Aplicar alguma pressão a pinça para segurar o útero para a agulha e expulsar aproximadamente 1 mL de BCM através do corno do útero. Nota: Tenha cuidado neste passo, como muita pressão na seringa causará um fluxo incontrolável de mídia, muitas vezes deixando o prato.</li>
		<li>Após a lavagem do útero, útero todo até 5 por prato de 60 mm, coloque o prato de volta na incubadora até coleção.</li>
	</ol>
</li>
	<li>
Colheita de embriões
	<ol>
<li>Usando uma pipeta de vidro estirado de mão e um aparelho de pipeta de boca, recolha blastocistos sob um escopo de dissecação, situado entre 25 X e 35 X. (Figura 1). Nota: Aparelho de pipeta boca consiste em 30-40 cm de tubo flexível, abaixo dos 5 mm identificação inserir um 1, 000 ponta da pipeta µ l, aponte primeiro em para uma extremidade e apor a pipeta de vidro nele. Adicione um filtro de seringa para a outra extremidade, com um anexo 1 cc seringa barril, sem atuador, para atuar como um pedaço da boca.</li>
		<li>Retirar pratos da incubadora e usando um padrão de grade, examinam cada parte do prato para embriões. Os embriões são escolhidos acima individualmente e coloque sobre a gota de BCM em um prato secundário.</li>
		<li>Após a coleta de todos os pratos, lavar os embriões mais uma vez em uma gota de BCM fresco e coloque no prato bem 4 para aguardar a injeção.</li>
	</ol>
</li>
</ol>4. o embrião injeção
<ol><li>Pegar as células ES individualmente com a agulha de injeção com base na confirmação.<ol>
<li>Preparar um prato de injeção pela adição de 5-7 mL de mídia de injeção (DMEM, 10% FBS e 20 mM HEPES) para um prato de injeção de fundo de vidro. As injeções são feitas à temperatura ambiente.</li>
		<li>Usando microinjector um ar ou óleo, aplica vácuo para a agulha de injeção para desenhar células ES no. Tome cuidado para manter-se como pouco espaço quanto possível entre as células. Conte as células ES aqui, ou na etapa de injeção.</li>
		<li>Selecione as células ES que são de médio a pequeno no tamanho em comparação com a população em geral de células, com uma superfície lisa e sem defeitos óbvios, tais como vacúolos. Nota: As populações de células ES variam grandemente em qualidade morfológica, e os padrões usados precisará ajustar com eles.</li>
	</ol>
</li>
	<li>Injete embriões com aproximadamente 15 células ES (Figura 1).<ol>
<li>Anexar uma pipeta de exploração dica para uma segunda microinjector de ar e posicionar um embrião perto da abertura do titular. Aplica vácuo para fixar o embrião para o titular.</li>
		<li>Ajuste para o eixo z movendo a pipeta de exploração cima ou para baixo para que o embrião é apenas tocar o slide, seguido por bem, focando a zona. Em seguida, ajuste o eixo Z da agulha de injeção para que as células ES perto da ponta estão em foco.</li>
		<li>Ajuste o embrião agora empurrando suavemente desde a agulha de injeção na parte superior ou inferior, girando-o para colocar o ICM na posição desejada.</li>
		<li>Insira a agulha de injeção através da zona e o ICM ou o trofoblasto, usando uma pressão firme, mas controlada. Exercendo uma ligeira pressão para o microinjector da agulha de injeção para ajudar a manter o blastocoel de entrar em colapso.</li>
		<li>Uma vez que a agulha penetrou o blastocoel, aplica pressão do microinjector para transferir células ES para o blastocoel do embrião. Pausar brevemente como o bisel da agulha começa a sair o embrião para permitir que a pressão no embrião para voltar ao normal, mantendo ainda as células. Nota: Referência a células ES grupo foram injetada normalmente, seguindo um tradicional ES celular injeção protocolo9, tomando cuidado para não tocar o ICM. Pilhas do ES também foram injetadas através do ICM, empurrando a agulha diretamente através do ICM (TICM) (Figura 2filme suplementar).</li>
	</ol>
</li>
</ol>5. transferência de eEmbryos
<ol><li>Transferência de embriões de camundongos Swiss Webster em E2.5, usando um dispositivo de transferência de embriões não-cirúrgico, por procedimentos de fabricação. Este procedimento não-cirúrgico requer sem anestesia ou cuidado especial no pós-operatório. Ratos de casa individualmente após a transferência de embriões e pela entrega e desmame subsequentes.</li>
</ol>

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	<li>Cong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+Genome+Engineering+Using+CRISPR/Cas+Systems.">Multiplex Genome Engineering Using CRISPR/Cas Systems.</a> Science. 339 (6), 819-823 (2013).</li><li>Mali, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-guided+human+genome+engineering+via+Cas9.">RNA-guided human genome engineering via Cas9.</a> Science. 339 (6), 823-826 (2013).</li><li>Friedel, R., Seisenberger, C., Kaloff, C., Wurst, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EUCOMM+the+European+Conditional+Mouse+Mutagenesis+Program.">EUCOMM the European Conditional Mouse Mutagenesis Program.</a> Brief Funct Genomic Proteomic. 6 (3), 180-185 (2007).</li><li>Austin, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+knockout+mouse+project.">The knockout mouse project.</a> Nat Genet. 36 (9), 921-924 (2004).</li><li>Coleman, J. L., Brennan, K., Ngo, T., Balaji, P., Graham, R. M., Smith, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Knockout+and+Reporter+Mouse+Line+Generation+and+Breeding+Colony+Establishment+Using+EUCOMM+Conditional-Ready+Embryonic+Stem+Cells:+A+Case+Study.">Rapid Knockout and Reporter Mouse Line Generation and Breeding Colony Establishment Using EUCOMM Conditional-Ready Embryonic Stem Cells: A Case Study.</a> Front Endocrinol (Lausanne). 30, 105 (2015).</li><li>Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W., Roder, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+completely+cell+culture-derived+mice+from+early-passage+embryonic+stem+cells.">Derivation of completely cell culture-derived mice from early-passage embryonic stem cells.</a> Proc Natl Acad Sci U S A. 90 (18), 8424-8428 (1993).</li><li>Eggan, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Male+and+female+mice+derived+from+the+same+embryonic+stem+cell+clone+by+tetraploid+embryo+complementation.">Male and female mice derived from the same embryonic stem cell clone by tetraploid embryo complementation.</a> Nat Biotechnol. 20 (5), 455-459 (2002).</li><li>Eggan, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+vigor,+fetal+overgrowth,+and+viability+of+mice+derived+by+nuclear+cloning+and+tetraploid+embryo+complementation.">Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation.</a> Proc Natl Acad Sci U S A. 98 (11), 6209-6214 (2001).</li><li>Eakin, G. 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Os autores têm sem interesses concorrentes para divulgar.

Isso tem sido pensado que qualquer perturbação do ICM pode levar à mortalidade, na verdade, protocolos atuais de microinjeção de blastocisto ainda alertam para este12,13. O que mostramos aqui é que microinjeção através do ICM não é prejudicial para o embrião e aumenta o rendimento de quimeras.
O mecanismo exato para este efeito não foi identificado. No entanto, a ruptura mecânica do ICM, e o hipoblasto que acompanha, deve...

Por quase 25 anos, a criação de camundongos geneticamente alvo tem sido um processo lento, muitas vezes prejudicado por um gargalo na fase de microinjeção de blastocistos ES celular para a geração de germline transmitindo quimeras. Recentes avanços, incluindo CRISPR/Cas91,2 direcionamento em pilhas do ES e elevado-throughput ES celulares consórcios de geração como KOMP, melhoraram a geração/disponibilidade de células ES geneticamente modificadas. No entanto, a geração de quimeras germline continua a ser um gargalo na criação de ratos geneticamente modificados destas células de ES3,4. Projetos de geração de célula do elevado-throughput ES tem sido atormentados por alta variabilidade na qualidade de célula ES, que é fundamental para a criação bem sucedida do germline quimeras. Além disso, algumas das linhas celulares ES comumente utilizadas são conhecidas por aneuploidia alta e difícil produção do germline quimeras competente5.
Muitos métodos têm sido desenvolvidos para a criação de ratos com uma quimera ou superior, ou completamente ES células derivadas ratos6,7,8,9,10. Enquanto cada um desses sistemas tem seus próprios méritos, eles também têm suas falhas. A geração de quimeras tetraploide, enquanto gerar células de ES 100% derivado de ratos, é ineficiente e geralmente limitado a linhas outbred 5,6. Novos métodos de injeção de mórula podem gerar alta porcentagem de quimeras, quase 100%, mas geralmente requerem equipamento adicional significativo (lasers ou piezos) e formação de10,11. Nos laboratórios onde estas técnicas já estão em vigor, esta nova metodologia pode não ser necessária.
O objetivo do presente estudo foi encontrar um método que usa técnicas comuns e equipamentos disponíveis para aumentar a taxa de geração de Quimera, aumentando a chance de transmissão do germline do alelo mutante para estabelecer a colônia de rato subsequentes.

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

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

Em um esforço para aumentar a eficiência na criação de ratos geneticamente modificados através de metodologias de célula ES, apresentamos uma adaptação para o atual protocolo de injeção de blastocisto. Aqui nós relatamos que uma simples rotação do embrião e injeção através de massa celular de Trans-interior (TICM) aumentou a porcentagem de ratos quiméricoes de 31% para 50%, sem nenhum equipamento adicional ou formação mais especializada. e mais uns 26 diferentes clones, e 35 totais clones foram injetados ao longo de um período de 9 meses. Não houve nenhuma diferença significativa entre as técnicas tradicionais de injeção e TICM na taxa de gravidez ou taxa de recuperação de embriões. Portanto, sem qualquer alteração importante no processo de injeção e um simples posicionamento do blastocisto e injetando através do ICM, liberando as células ES na cavidade blastocoel pode potencialmente aumentar a quantidade de produção quimérico e subsequente transmissão da linha germinal.

Tratamento de linha celular como o efeito aleatório, um modelo linear de efeitos mistos foi usado para analisar os dados no software (por exemplo, SAS (versão 9.2)). Cada análise controlada para o número de embriões transferidos e a linha celular. A segunda análise também controlada para o número de filhotes que sobreviveram.
Para este estudo, cada linha celular do ES e o clone foi injetado em ambos com tradicionais e t?...

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

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

Aqui nós apresentamos um protocolo para aumentar a produção da Quimera sem o uso de novos equipamentos. Uma mudança de orientação simples do embrião para injeção pode aumentar o número de embriões produzidos e potencialmente reduzir a linha do tempo de transmissão da linha germinal.

Trans-interno pilha injeção maciça de células-tronco embrionárias leva a taxas mais elevadas de quimerismo

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

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Biology

10.2K visualizações.  NIEHS/NIH. Aqui nós apresentamos um protocolo para aumentar a produção da Quimera sem o uso de novos equipamentos. Uma mudança de orientação simples do embrião para injeção pode aumentar o número de embriões produzidos e potencialmente reduzir a linha do tempo de transmissão da linha germinal.

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

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

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

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

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

Ratos Transnuclear com pré-definidos T Especificidades receptor da célula contra a Toxoplasma gondii Obtidos Via TNCS

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

Imunologia e Infecção

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

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

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

Métodos de Transferência Precisamente localizada de células ou DNA em adiantado postimplantation embriões de camundongos

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

Biologia do Desenvolvimento

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

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

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

Combinacional tratamento de tricostatina A e a vitamina C melhora a eficiência da clonagem de ratos por transferência Nuclear de células somáticas

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

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

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

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

Tecido alvo quimeras embrionárias: Zebrafish celular Gástrula Transplante

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

Biologia

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

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

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

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

Intravenosa e Intra-amniótica no Utero transplante no modelo murino

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

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

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

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

Sistema baseado em microinjeção para na implantação de Vivo do Cardiomyocytes embrionárias no embrião aviária

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

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

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

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

Dechorionation de Embriões Medaka e Transplante de Células de Geração de Quimeras

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

Xenotransplantation of Human Stem Cells into the Chicken Embryo

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

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

Xenotransplante de células-tronco humanas no embrião de galinha

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

Single Cell Transfection in Chick Embryos

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

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

Transfecção única célula em embriões de galinha

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

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

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

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

Direcionamento de genes altamente eficientes mediados por CRISPR/Cas9 em células-tronco embrionárias para o desenvolvimento de modelos de camundongos manipulados por genes

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