A colheita de medula óssea de fêmures e tíbias <ol><li> O primeiro passo do procedimento é a colheita de medula óssea de fêmures e tíbias o de um 6 - a 12 semanas de mouse antigo (que estamos usando camundongos BALB / c, mas você pode fazer este procedimento com qualquer tensão mouse). Para começar, euthanize o mouse por inalação de CO 2 (não irá ser exibido). Fêmur e tíbia colheita das pernas traseiras de cada um. Então, com uma lâmina de barbear, cortar cada extremidade do fémur para remover o quadril e joelho e para expor a medula. </li><li> De forma semelhante, corte cada extremidade da tíbia para remover o joelho eo tornozelo regiões adjacentes e para expor a medula. </li><li> Pegue uma seringa de 3 mL com uma agulha de calibre 26 e preenchê-lo com RPMI suplementado com SFB 10%, penicilina, estreptomicina e beta-mercaptoetanol (BME = 100 uM). Usando uma pinça, segure o osso por uma placa de Petri contendo RPMI mídia. Inserir a agulha da seringa em uma das extremidades do osso e pressionar o êmbolo para expulsar da medula óssea. A agulha da seringa pode ser movida para cima e para baixo dentro do osso para expulsar medula residual. </li><li> Repita o procedimento para todos os ossos, e então proceder com o estabelecimento das culturas. </li></ol> Estabelecer culturas <ol><li> Para estabelecer as culturas de medula óssea, pipetar a medula óssea vermelha para cima e para baixo várias vezes para quebrar o tecido em uma suspensão de células. </li><li> Colocar um filtro de 70 micron em cima de um tubo cônico de 50 mL, e adicione a suspensão de células para o filtro. </li><li> Lavar a placa de Petri uma vez com RPMI, em seguida, adicione a água para o filtro 70 mícron. </li><li> Usando o final de borracha de uma seringa de 1 mL êmbolo, moer os pedaços restantes de medula óssea na parte superior do filtro micron 70. </li><li> Lavar o filtro com um tempo RPMI. </li><li> Depois de lavar o filtro, centrifugar a suspensão celular a 1200 rpm por 5 minutos. </li><li> Lave o pellet celular pela ressuspensão em PBS. </li><li> Contar as células usando um hemocitômetro. Normalmente, obter a 90 milhões de células de ossos um rato (dois fêmures e duas tíbias). Após a contagem das células, centrifugar novamente a 1200 rpm por 5 minutos. </li><li> Ressuspender as células em uma pequena quantidade de RPMI e alíquota-los em frascos de cultura de tecidos contendo RPMI suficiente para que a concentração final é de 1 milhão de células por ml. </li><li> Adicionar citocinas para promover o desenvolvimento do seu tipo de célula de interesse. Neste caso, a citocina interleucina-3 (IL-3 = 10 ng / mL) é adicionado para promover o desenvolvimento de basófilos e mastócitos. Para adquirir basófilos, incubar por 10 dias. Para mastócitos, incubar por 5 semanas. Ao gerar mastócitos, a mídia e IL-3 deve ser trocada uma vez por semana. </li><li> Aqui está uma olhada em como mastócitos deve aparecer apenas após o plaqueamento. Esta imagem demonstra a diferenciação de células da medula mastro em basófilos e mastócitos 10 dias após a adição de interleucina-3. </li><li> Uma vez que o tipo de célula desejada é obtida, prossiga com o passo eletroporação. </li></ol> Células Electroporating <ol><li> Para começar a etapa de eletroporação, determinar o número de células no frasco de cultura. </li><li> As células são tipicamente electroporated a uma densidade de 10 milhões por mL, então transferir o número de células necessárias para tubos cônicos e centrifugar os tubos a 1200 rpm por 5 minutos. </li><li> Após a centrifugação, aspirar a mídia, lavar as células com PBS 1X, e centrifugar novamente a 1200 rpm por 5 minutos. </li><li> Aspirar o PBS e adicionar Bio-Rad Gene tampão de eletroporação Pulser para fazer uma suspensão de células de 10 milhões de células por mL. </li><li> Depois de ressuspender as células, adicionar o DNA desejado plasmídeo em uma concentração final de 10 a 20 microgramas por mL. </li><li> Alíquota de 150 microlitros da suspensão de células em poços de sua escolha em uma placa de eletroporação de 96 poços. </li><li> Coloque a placa na câmara de eletroporação placa MXcell e feche a tampa. </li><li> Antes de transfecção de mastócitos, um protocolo de eletroporação deve ser programado no MXcell menos usando um protocolo preestabelecido ou armazenados. Através de experimentos de otimização descobrimos que a mais alta eficiência de transfecção de mastócitos ocorrem usando protocolos de pulso de onda quadrada. Vamos alterar as condições de eletroporação na placa para entregar 300V/20ms, 350/15ms, 350V/20ms, e pulsos de onda quadrada na 350V/10ms 2000uF e 1000 ohms. Uma vez que o protocolo foi definido e carregado para o dispositivo, pressione &quot;Pulse&quot; para electroporate as células. </li><li> Depois de eletroporação é completo, a transferência das células para uma placa de cultura de tecidos. Normalmente, a transferência de cada amostra de 150 microlitros eletroporação a um poço em uma placa de cultura de 48 poços contendo 300 microlitros tecido RPMI (suplementado com 10% FCS, penicilina, estreptomicina e beta-mercaptoetanol) com 10 nanogramas por ml de interleucina-3. As células são incubadas overnight a 37 ° C, em seguida, analisadas 24 horas depois de expressão e supressão. </li></ol> TranResultados sfection A imagem de microscopia fluorescente das células após a eletroporação bem sucedido usando 20 microgramas por mL GFP plasmídeo é mostrado no vídeo. Usando o eletroporação MXcell a eficácia do sistema de transfecção de cerca de 30% pode ser obtida. O sistema permite variar as condições para maximizar a eficiência de transfecção, mantendo a viabilidade das células.

À medida que mais informação genômica emerge e novas ferramentas, como siRNA são desenvolvidos, o uso de células fisiologicamente relevante se tornar cada vez mais importante para promover nossa compreensão das vias de doença, interações proteína-proteína, e transdução de sinal. As células primárias são obtidas diretamente a partir de tecidos ou fluidos e cultivadas in vitro. Estas células podem ser manipuladas em uma série de maneiras, incluindo através da introdução de material genético exógeno. É cada vez mais evidente que a maneira mais eficaz para introduzir DNA plasmídeo ou siRNA em células primário é eletroporação. Eletroporação expõe as células a pulsos elétricos, a fim de aumentar a permeabilidade transitória da membrana celular, permitindo assim exógenos ácidos nucléicos para entrar na célula. Este método de transfecção pode ser otimizada para acomodar as diferenças entre os diferentes tipos de células / linhas e, portanto, pode ser usado para transfecção mastócitos, bem como de qualquer outro osso de células derivadas da medula. Além disso, ao contrário viral mediada transfecção, eletroporação não impõe limites no tamanho do DNA transfectado, nem requerem uma preparação extensiva. Em contraste com lipídica mediada por transfecção, eletroporação pode ser menos tóxico e não resultar em aprisionamento endossomal do ácido nucléico transfectadas. Bio-Rad foi desenvolvido o sistema de eletroporação MXcell especificamente para estas células. o MXcell permite variar as condições para maximizar sua eficiência de transfecção e viabilidade celular. A fim de otimizar a eficiência de transfecção e minimizar a morte celular, uma multiplicidade de parâmetros eletroporação incluindo tensão, capacitância, resistência e comprimento do pulso pode ser ajustada e avaliados.

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the preparation of primary hematopoietic cell cultures from murine bone marrow for electroporation

É cada vez mais evidente que eletroporação é a forma mais eficaz para introduzir DNA plasmídeo ou siRNA em células primárias. O Gene Pulser sistema de eletroporação MXcell e Gene tampão de eletroporação Pulser foram desenvolvidos especificamente para transfecção ácidos nucleicos em células de mamíferos e de difícil transfecção de células, tais como vídeo primário e cells.This-tronco demonstra como estabelecer culturas primárias de células hematopoiéticas da medula óssea de camundongos , e depois prepará-los para eletroporação no sistema MXcell. Começamos isolando fêmur e tíbia. Medula óssea de ambos os fêmur ea tíbia são, então, colhidas e culturas são estabelecidas. Células da medula óssea cultivadas são, então, transfectadas e analisados.

Watch this Scientific Journal Video about The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation at JoVE.com

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

Este procedimento descreve como estabelecer culturas primárias de células hematopoiéticas da medula óssea de camundongos e é seguido por transfecção usando o Gene Pulser sistema de eletroporação MXCell.

Elaboração de culturas de células primárias hematopoéticas de medula óssea de camundongos para Eletroporação

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

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Biology

25.7K visualizações.  Bio-Rad Laboratories, Inc.  ChineseVersion 

Vídeo: The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

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 derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

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 ...

Biologia

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

Culturas primárias neuronais do cérebro de Tarde Stage Drosophila pupas

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Preparação de culturas de neurônios a partir de embriões Midgastrula Stage Drosophila

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

Eletroporação de Micobactérias

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

Cultura de células dendríticas mielóides a partir de precursores da medula óssea

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

Principal Dissociated Midbrain dopamina culturas de células de recém-nascidos de roedores

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

Homing of Hematopoietic Cells to the Bone Marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

Homing das células hematopoiéticas da medula óssea

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

Cultura primária e Eletroporação Plasmid do Órgão de Corti murino.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Culturas de células primárias de Drosophila Os embriões Gástrula

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

Preparação de Primário Miogênica de Células Precursoras / Culturas de mioblastos de Vertebrados basais Linhagens

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...