Name: trans-inner cell mass injection of embryonic stem cells leads to higher chimerism rates
Uploaded: 2018-05-29T13:00:00
Description: Watch this Scientific Journal Video about Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates at JoVE.com

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...

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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+knockout+mouse+project.">The knockout mouse project.</a> Nat Genet. 36 (9), 921-924 (2004).</li><li>Coleman, J. L., Brennan, K., Ngo, T., Balaji, P., Graham, R. M., Smith, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Knockout+and+Reporter+Mouse+Line+Generation+and+Breeding+Colony+Establishment+Using+EUCOMM+Conditional-Ready+Embryonic+Stem+Cells:+A+Case+Study.">Rapid Knockout and Reporter Mouse Line Generation and Breeding Colony Establishment Using EUCOMM Conditional-Ready Embryonic Stem Cells: A Case Study.</a> Front Endocrinol (Lausanne). 30, 105 (2015).</li><li>Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W., Roder, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+completely+cell+culture-derived+mice+from+early-passage+embryonic+stem+cells.">Derivation of completely cell culture-derived mice from early-passage embryonic stem cells.</a> Proc Natl Acad Sci U S A. 90 (18), 8424-8428 (1993).</li><li>Eggan, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Male+and+female+mice+derived+from+the+same+embryonic+stem+cell+clone+by+tetraploid+embryo+complementation.">Male and female mice derived from the same embryonic stem cell clone by tetraploid embryo complementation.</a> Nat Biotechnol. 20 (5), 455-459 (2002).</li><li>Eggan, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+vigor,+fetal+overgrowth,+and+viability+of+mice+derived+by+nuclear+cloning+and+tetraploid+embryo+complementation.">Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation.</a> Proc Natl Acad Sci U S A. 98 (11), 6209-6214 (2001).</li><li>Eakin, G. S., Hadjantonakis, A. K., Papaioannou, V. E., Behringer, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+potential+and+behavior+of+tetraploid+cells+in+the+mouse+embryo.">Developmental potential and behavior of tetraploid cells in the mouse embryo.</a> Dev Biol. 288 (1), 150-159 (2005).</li><li>Huang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+production+of+mice+from+embryonic+stem+cells+injected+into+four-+or+eight-cell+embryos+by+piezo+micromanipulation.">Efficient production of mice from embryonic stem cells injected into four- or eight-cell embryos by piezo micromanipulation.</a> Stem Cells. 26 (7), 1883-1890 (2008).</li><li>Poueymirou, W. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=F0+generation+mice+fully+derived+from+gene-targeted+embryonic+stem+cells+allowing+immediate+phenotypic+analyses.">F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses.</a> Nat Biotechnol. 25 (1), 91-99 (2007).</li><li>Behringer, R., Gertsenstein, M., Nagy, K. V., Nagy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the Mouse embryo. , (2014).</li><li>Pease, S., Saunders, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advanced Protocols for Animal Transgenesis. , (2011).</li><li>Young, M. 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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>b-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&#60;c-2J&#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 ...

The overall goal of this modified procedure is to increase chimeric production without incurring additional costs or long training periods. This method can help laboratories become more efficient in chimera production, especially in cases where the ES cells are less than optimal. The main advantage of this technique is that it requires no additional equipment and no specialized training.Demonstrating the procedure today will be Thomas Hagler. He's a biologist and graduate student who works in the core lab. To harvest the embryos, blot the uterus on a lab tissue to remove the excess blood and place the organ in a new 60 millimeter dish under a low magnification dissecting microscope.Using dissecting forceps to carefully and gently hold the uterus near the oviduct, insert a 25 gauge needle attached to a 10 milliliter syringe containing 10 milliliters of blast collection medium into the center of the uterus just beyond the tips of the forceps in as parallel a plane as possible. Apply pressure to the forceps to secure the needle within the uterus and gently flush approximately one milliliter of medium through the uterine horn. When all of the blastocysts have been expelled, assemble the mouth pipette apparatus and use the mouth pipette to transfer the individual embryos into single drops of blast collection medium in a second 60 millimeter dish.Then wash each embryo in a second drop of blast collection medium and transfer the embryos into a four-well dish until their injection. To begin, use a microinjector to apply vacuum to the injection needle. Place a dish of ES cells under the injection microscope where the injection needle and embryo holder pipette are attached.It is essential to the success of the injection to take the time to properly setup the microscope. This includes the proper amount of media in the needle and the correct alignment of the z-axis. Use a microinjector to apply vacuum to the injection needle.Keeping as little space between the cells as possible, draw in smoothly surfaced small to medium sized embryonic cells without obvious defects from the dish of embryos. After collecting at least 15 cells, position an embryo near the opening of the holder. Apply vacuum to secure the embryo to the holder and move the holding pipette up or down to adjust the z-axis until the embryo is just touching the slide.Fine focus on the zone of pellucida and adjust the z-axis of the injection needle until the embryonic cells near the tip of the needle are in focus. If needed, use the injection needle to gently roll the blastocyst until the inner cell mass is in the appropriate position for injection. Using a firm but controlled push, insert the injection needle through the zone of pellucida into the inner cell mass and apply a slight pressure to the microinjector of the injection needle to help keep the blastocoele from collapsing.Once the needle has penetrated the blastocoele, apply pressure to the microinjector to transfer all of the embryonic stem cells into embryo, pausing briefly as the bevel of the needle starts to exit the embryo to allow the pressure in the embryo to return to normal while still retaining the cells. When all of the blastocysts have been injected, use a nonsurgical embryo transfer device to deliver the embryos to embryonic stage 2.5 Swiss Webster mice according to the manufacturer's instructions and house the mice individually after each transfer through delivery and subsequent weaning. In this representative experiment, the pregnancy rates were unaltered between the trans inner cell mass injected and control traditionally injected groups and an equal number of pups survived to weaning from each group.Notably, while the number of mice at weaning remained unchanged, a significantly higher number of pups from the trans inner cell mass injected group displayed coat-color chimerism compared to the control injected group. The best part of this technique is that it only requires a simple change in the orientation of the embryo to facilitate greater production in chimeras. Proper setup of the microscope is essential.If the microscope and manipulators aren't setup properly and maintained well, the injection process will not work. If you're new to embryo injections, repetition is the only way to learn. For beginners, embryo handling tends to be the most difficult part of the procedure.Being able to move embryos efficiently from one dish to another without losing any is the most crucial part of the experiment. After watching this video, you should have a good understanding of how a simple change to your standard injection technique can help you become more efficient in chimera production.

Research

JoVE Journal

Biology

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Propagação de-tronco embrionárias humanas (ES) Células

Biologia

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

Derivação de Células-Tronco Hematopoéticas murino de Células-Tronco Embrionárias

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

Derivação de células estaminais embrionárias humanas por Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Embrionárias com células-tronco derivadas de células endoteliais para tratamento da isquemia dos membros posteriores

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

Isolamento e derivação de células embrionárias de rato Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Diferenciação de células-tronco embrionárias em precursores oligodendroglial

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Mapeamento de destino de células estaminais embrionárias humanas por formação de teratomas

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

Geração robusto de Hepatócito-like Células de populações humanas células-tronco embrionárias

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Uso de Lyso Tracker para detectar a morte celular programada em embriões e diferenciação das células-tronco embrionárias

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Indução e Análise de epitelial para mesenquimal Transição

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...