Este trabajo fue financiado por becas CIRM # TR-1250, RT1-01108, y 00502-CL1.

Nota: Todos los procedimientos con animales debe ser aprobado por el IACUC o equivalente. Todo el equipo quirúrgico debe ser esterilizado antes de la cirugía. Guantes estériles, las cortinas de gasa y se debe utilizar. 1. Preparación antes de la cirugía <ol><li> Obtenga 6 semanas de edad Mus musculus CbySmn.CB17-Prkdc SCID / J ratones machos o otra cepa de ratón inmunodeficiente. </li><li> Esterilice todos los instrumentos quirúrgicos, guantes y gasa. </li><li> Disociar hPSCs ser inyectados con accutase. </li><li> Contar las células y resuspender 1.000.000 células por 20-30 l en matrigel diluido 1:1 en DMEM/F-12. Mantener las células en hielo hasta que termine. </li><li> Tenga en cuenta que a menudo es útil hacer una carrera a través del experimento en el que se inyecta azul de tripano. Esto ayudará a identificar cualquier problema potencial antes de que las células se pierden. </li><li> En el vivero, anestesiar el puntero del ratón de acuerdo con los procedimientos aceptados en su institución. En nuestros experimentos, hemos utilizado un AnesthEIAS de la máquina con isoflurano. Los ratones fueron puestos inicialmente en una cámara de inducción con el oxígeno 1L/min y 3-4% isoflurano. Una vez anestesiado, un cono de la nariz con el oxígeno 1L/min e isoflurano al 2-3% se utilizó. </li><li> El abdomen afeitado con maquinilla, y limpiar la pared anterior del abdomen del ratón, comenzando desde el centro del abdomen y de trabajo en sentido horario hacia el exterior. El primer uso solución de povidona yodada y luego lave con etanol al 70%. Repita 3 veces, el cambio de hisopos en cada ocasión. Traslado del animal a una compresa caliente para mantener al animal caliente dentro de un cultivo de tejido o una campana de disección. </li><li> Hacer una incisión de 1 cm longitudinal a través de la piel y el peritoneo con tijeras quirúrgicas estériles justo por debajo del nivel de la articulación de la cadera. </li><li> Mientras mantiene el peritoneo con una pinza, llegar hacia abajo, hacia la cadera derecha con otro forcept estéril y extraiga el tejido adiposo blanco, junto con los testículos adjuntos. </li><li> Coloque los testículos en una gasa estéril. </li><li> Llene una tuberculina o Hamilton (1 cc) jeringa de wiª los hPSCs a ser inyectado. Tenga en cuenta que es una buena idea incluir una línea de control hPSC que sabe que es pluripotente como WA09 (también conocido como H9). De esta manera usted tiene un control positivo que se puede utilizar para determinar si la inyección o la cirugía era defectuoso. </li><li> Lentamente inyectar los hPSCs (20-30 l) en el centro de la cápsula testículo lejos de cualquier principales vasos sanguíneos, parando si la cápsula testicular comienza a hincharse. </li><li> Retirar la aguja lentamente para evitar el reflujo de las células. </li><li> Con unas pinzas, transfiera los testículos y la espalda tejido graso a su posición original en el abdomen. </li><li> Cierre del peritoneo con 2 o 3 puntos de sutura reabsorbibles y cerrar la piel con autoclips. </li><li> El ratón se debe mantener caliente hasta que se recupere y teniendo en cuenta algún tipo de analgésico (ver lo que es aceptado en el vivero) después de la cirugía dos veces al día durante 1-2 días. Nótese que ni el anestésico ni el analgésico con interferir con el desarrollo del tumor. </li><li> Monitorear ºe animal para el crecimiento del tumor durante 6-12 semanas. Muy pocas veces, los tumores pueden crecer a 5 mm de tamaño anteriores a 6 semanas después de la inyección, por lo tanto es importante controlar los animales. Si esto sucede, los animales deben ser sacrificados y procesados ​​y analizados los tumores como de costumbre. </li><li> Una vez que el tumor es palpable y llega a aproximadamente 5 mm de tamaño, anestesiar el ratón y el sacrificio de acuerdo con los procedimientos aceptados animales de protocolo. </li><li> Extirpar el tumor y el documento de acuerdo - Una fotografía tamaño, medida, peso. </li><li> Cortar en trozos pequeños tumores y fijar en la solución de paraformaldehído al 4%. Almacenar en paraformaldehído al 4% hasta que las muestras se envían a un patólogo para su corte, coloración y el análisis. </li></ol> 2. Resultados representativos: Cuando este protocolo se realiza como se ha descrito y la línea celular inyectado es pluripotentes, un palpable, visualmente evidente, tumor debe formar un plazo de 12 semanas a lo sumo. Para las líneas establecidas hPSC como WA09, vemos típicamentelos tumores dentro de 6 semanas. Para las líneas de IPSC no es raro ver a los tumores dentro de las 8-10 semanas. Es muy importante para inyectar ratones con una línea que es conocido por ser pluripotentes, como un control positivo, a fin de estar seguro de que el procedimiento se realizó correctamente. Los tumores por lo general un aspecto muy heterogéneo y tienen muchos quistes conectados (Figura 1). El análisis de las muestras tumorales de un patólogo debe mostrar los tejidos diferenciados de las tres capas germinales (Figura 2). <img alt="Figura 1" src="/files/ftp_upload/3177/3177fig1.jpg" /> Figura 1. Típica teratoma hPSC deriva en la cápsula del testículo. Siguiendo el protocolo descrito, un millón de WA09 células se inyectaron en la cápsula testículo de un ratón inmune comprometido. Seis semanas más tarde se observó un teratoma. A) Teratoma retirado del ratón. B) Acercar la imagen del teratoma. Tenga en cuenta la heterogeneidad de las estructuras y el quiste. <img alt="Figura 2" src = &quot;/ files/ftp_upload/3177/3177fig2.jpg&quot; /&gt; Figura 2. Hematoxilina y eosina tiñen secciones del teratoma muestran los tejidos de cada capa germinal. Tras la fijación, el teratoma era seccionadas y teñidas con hematoxilina y eosina. Análisis por un patólogo reveló la presencia de células de cada una de las 3 capas germinales.

<ol>
	<li>Bradley, A., Evans, M., Kaufman, M. H., Robertson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+germ-line+chimaeras+from+embryo-derived+teratocarcinoma+cell+lines.">Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines.</a> Nature. 309, 255-255 (1984).</li><li>Evans, M. J., Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+in+culture+of+pluripotential+cells+from+mouse+embryos.">Establishment in culture of pluripotential cells from mouse embryos.</a> Nature. 292, 154 (1981).</li><li>Martin, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+pluripotent+cell+line+from+early+mouse+embryos+cultured+in+medium+conditioned+by+teratocarcinoma+stem+cells.">Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.</a> Proc. Natl. Acad. Sci. U. S. A. 78, 7634-7634 (1981).</li><li>Downing, G. J., Battey, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+assessment+of+the+first+20+years+of+research+using+mouse+embryonic+stem+cell+lines.">Technical assessment of the first 20 years of research using mouse embryonic stem cell lines.</a> Stem Cells. 22, 1168-1168 (2004).</li><li>Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1145 (1998).</li><li>Reubinoff, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+from+human+blastocysts:+somatic+differentiation+in+vitro.">Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro.</a> Nat Biotechnol. 18, (2000).</li><li>Muller, F. J., Goldmann, J., Loser, P., Loring, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+call+to+standardize+teratoma+assays+used+to+define+human+pluripotent+cell+lines.">A call to standardize teratoma assays used to define human pluripotent cell lines.</a> Cell Stem Cell. 6, 412-412 (2010).</li><li>Brivanlou, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells.+Setting+standards+for+human+embryonic+stem+cells.">Stem cells. Setting standards for human embryonic stem cells.</a> Science. 300, 913-913 (2003).</li><li>Lensch, M. W., Schlaeger, T. M., Zon, L. I., Daley, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Teratoma+formation+assays+with+human+embryonic+stem+cells:+a+rationale+for+one+type+of+human-animal+chimera.">Teratoma formation assays with human embryonic stem cells: a rationale for one type of human-animal chimera.</a> Cell Stem Cell. 1, 253-25 (2007).</li><li>Gertow, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organized+development+from+human+embryonic+stem+cells+after+injection+into+immunodeficient+mice.">Organized development from human embryonic stem cells after injection into immunodeficient mice.</a> Stem. Cells. Dev. 13, 421-421 (2004).</li><li>Gertow, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+embryonic+stem+cell-derived+teratomas+for+the+assessment+of+pluripotency.">Isolation of human embryonic stem cell-derived teratomas for the assessment of pluripotency.</a> Curr Protoc Stem Cell Biol. Chapter 1, Unit1B 4-Unit1B 4 (2007).</li></ol>

No hay conflictos de interés declarado.

El método que aquí se presenta ofrece una alta fiabilidad, los medios directos de la generación de los teratomas de hPSCs en la cápsula del testículo. Hay varios parámetros críticos en esta técnica. En particular, es importante para inyectar líneas hPSC celulares que se sabe que son pluripotentes como control. Otros parámetros importantes incluyen el intervalo de tiempo entre la inyección y la observación del tumor. Para las líneas celulares como WA09, los teratomas se deben observar en 6-8 semanas. Para las nuevas líneas de IPSC nos encontramos con que a menudo se requieren 10 semanas. Otra preocupación es el número de células inyectadas. Se inyecta un millón de células, pero el ensayo se puede hacer fácilmente con un menor número de células. Además, el medio de inyección es importante. Se encuentra que se obtienen los mejores resultados cuando las células se inyectan en una mezcla 1:1 de Matrigel y DMEM/F-12, en oposición a PBS o DMEM/F-12.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Accutase </td><td> Invitrogen </td><td> A1110501 </td></tr><tr><td> DMEM/F-12 </td><td> Invitrogen </td><td> 113300-032 </td></tr><tr><td> Matrigel </td><td> BD Biosciences </td><td> 354277 </td></tr></tbody></table>

teratoma generation in the testis capsule

Las células madre pluripotentes (PSC) tiene la característica única que pueden diferenciarse en células de las tres capas germinales. Esto los convierte en una herramienta potencialmente valiosa para el tratamiento de muchas enfermedades diferentes. Con la llegada de células madre pluripotentes inducidas (iPSCs) y la investigación continúa con células madre embrionarias humanas (hESCs) es necesario para los ensayos que puedan demostrar que una línea celular particular es pluripotente. Transmisión de línea germinal ha sido el estándar de oro para demostrar la pluripotence de células madre embrionarias de ratón (MESC) líneas 1,2,3. El uso de este ensayo, los investigadores pueden demostrar que una línea MESC puede hacer todos los tipos de células en el embrión incluidas las células germinales 4. Con la generación de líneas de CES 5,6 humana, el ensayo apropiado para demostrar pluripotence de estas células estaba claro desde el CES humanos no pueden ser probados para la transmisión de línea germinal. Como sustituto, el ensayo de teratoma se utiliza actualmente para demostrar la pluripottransparencia de las células madre pluripotentes (hPSCs) 7,8,9. Aunque este ensayo recientemente ha sido objeto de escrutinio y las nuevas tecnologías se están explorando activamente, el ensayo de teratoma es el actual estándar de oro 7. En este ensayo, las células en cuestión se inyectan en un ratón inmune comprometido. Si las células son pluripotentes, un teratoma eventualmente desarrollar y secciones del tumor se muestran los tejidos de las 3 capas germinales 10. En el ensayo de teratoma, hPSCs se puede inyectar en las diferentes áreas del ratón. Los sitios de inyección más comunes incluyen la cápsula testículo, la cápsula del riñón, hígado, o en la pata tanto por vía subcutánea o intramuscular 11. Aquí se describe un protocolo robusto para la generación de teratomas de hPSCs utilizando la cápsula testículo como el sitio para el crecimiento del tumor.

Watch this Scientific Journal Video about Teratoma Generation in the Testis Capsule at JoVE.com

Teratoma Generation in the Testis Capsule

Las células madre pluripotentes (hPSCs) tienen el potencial para tratar una gran variedad de enfermedades diferentes. La utilidad de estas células reside en el hecho de que pueden diferenciarse en cualquier tipo de célula en el cuerpo. Aquí se describe el ensayo de teratoma, que se utiliza para demostrar la pluripotence de hPSCs.

Teratoma generación en la cápsula del testículo

Pluripotent stem cells (PSCs) have the unique characteristic that they can differentiate into cells from all three germ layers.  This makes them a ...

teratoma-generation-in-the-testis-capsule

Research

JoVE Journal

Medicine

20.6K vistas.  Scripps Research Institute. Las células madre pluripotentes (hPSCs) tienen el potencial para tratar una gran variedad de enfermedades diferentes. La utilidad de estas células reside en el hecho de que pueden diferenciarse en cualquier tipo de célula en el cuerpo. Aquí se describe el ensayo de teratoma, que se utiliza para demostrar la pluripotence de hPSCs.

Video: Teratoma Generation in the Testis Capsule

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

Una estrategia para identificar mutaciones de novo en los trastornos comunes, tales como el autismo y la esquizofrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Investigación

Medicina

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

Aspectos técnicos de la aortocava Fístula Ratón

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.

We describe the manufacture of compressed hormone pellets, as well as subcutaneous surgical implantation into mice. This strategy can be combined with the growth of cell and tissue xenografts under the renal capsule of athymic nude mice to evaluate hormonal carcinogenesis and regulation of benign prostate growth.

Renal Cápsula xenotrasplante y subcutánea Pellet Implantación para la Evaluación de la próstata Carcinogénesis y la hiperplasia prostática benigna

New therapies for two common prostate diseases,  prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend  critically on experiments ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

Generación y cuantificación de 3 dimensiones de las lesiones arteriales en ratones mediante tomografía óptica de proyección

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

Recolección y extracción de ADN de la saliva de Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment, their rarity and the overwhelming excess of hematopoietic cells. Towards this end, we developed an in vitro 2D clonogenic model of dormancy of estrogen-sensitive breast cancer cells in the bone marrow. The model consists of a few key elements necessary for dormancy. These include 1) the use of estrogen sensitive breast cancer cells, which are the type likely to remain dormant for extended periods, 2) incubation of cells at clonogenic density, where the structural interaction of each cell is primarily with the substratum, 3) fibronectin, a key structural element of the marrow and 4) FGF-2, a growth factor abundantly synthesized by bone marrow stromal cells and heavily deposited in the extracellular matrix. Cells incubated with FGF-2 form dormant clones after 6 days, which consist of 12 or less cells that have a distinct flat appearance, are significantly larger and more spread out than growing cells and have large cytoplasm to nucleus ratios. In contrast, cells incubated without FGF-2 form primarily growing colonies consisting of >30 relatively small cells. Perturbations of the system with antibodies, inhibitors, peptides or nucleic acids on day 3 after incubation can significantly affect various phenotypic and molecular aspects of the dormant cells at 6 days and can be used to assess the roles of membrane-localized or intracellular molecules, factors or signaling pathways on the dormant state or survival of dormant cells. While recognizing the in vitro nature of the assay, it can function as a highly useful tool to glean significant information about the molecular mechanisms necessary for establishment and survival of dormant cells. This data can be used to generate hypotheses to be tested in vivo models.

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo.

Un<em&gt; In Vitro</em&gt; Latencia Modelo de cáncer de mama estrógeno-sensibles en la médula ósea: una herramienta de Estudios mecanismo molecular y Hipótesis Generación

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant ...

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs&#160;that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d&#160;with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Here we report a rapid and efficient gene editing method based on RMCE in the AAVS1 locus of human Pluripotent Stem Cells (hPSCs) that improves upon previously described systems. Using this technique, isogenic lines can be rapidly and reliably generated for proper comparative studies, facilitating transgenesis-mediated research with hPSCs.

Generación rápida y eficiente de las células madre pluripotentes recombinante humana mediada por la recombinasa Cambio en el cassette<em&gt; AAVS1</em&gt; Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is ...

Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The goal of computational methods for drug repurposing is to enable solutions for safer, cheaper and faster drug discovery. Towards this end, we developed a novel method that integrates genetic and clinical phenotype data from large-scale GWAS and PheWAS studies with detailed drug information on the concept of transitive Drug-Gene-Disease triads. We created "RE:fine Drugs," a freely available, interactive dashboard that automates gene, disease and drug-based searches to identify drug repurposing candidates. This web-based tool supports a user-friendly interface that includes an array of advanced search and export options. Results can be prioritized in a variety of ways, including but not limited to, biomedical literature support, strength and statistical significance of GWAS and/or PheWAS associations, disease indications and molecular drug targets. Here we provide a protocol that illustrates the functionalities available in the "RE:fine Drugs" system and explores the different advanced options through a case study.

Drug Repurposing Hypothesis Generation Using the &quot;RE:fine Drugs&quot; System

Here we describe a protocol using the web-based drug repurposing hypothesis generation tool: "RE:fine Drugs." This protocol can be modified to a user's preferences at the level of the query type (gene, drug or disease) and/or the range of available advanced options.

Reutilización de drogas Hipótesis Generación Usando el &quot;RE: Drogas finas&quot; Sistema

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

La generación de fracturas femorales cerradas en ratones: un modelo para el estudio del hueso curativo

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

Generation of Porcine Testicular Organoids with Testis Specific Architecture using Microwell Culture

Organoids are three dimensional structures composed of multiple cell types that are capable of recapitulating tissue architecture and functions of organs in vivo. Formation of organoids has opened up different avenues of basic and translational research. In recent years, testicular organoids have garnered interest in the field of male reproductive biology. Testicular organoids allow for the study of cell-cell interactions, tissue development, and the germ cell niche microenvironment and facilitate high throughput drug and toxicity screening. A method is needed to reliably and reproducibly generate testicular organoids with testis specific tissue architecture. The microwell culture system contains a dense array of pyramid-shaped microwells. Testicular cells derived from pre-pubertal testes are centrifuged into these microwells and cultured to generate testicular organoids with testis-specific tissue architecture and cell associations. Thousands of homogeneous organoids can be generated via this process. The protocol reported here will be of broad interest to researchers studying male reproduction.

Here, we present a protocol for the reproducible generation of porcine testicular organoids with testis specific tissue architecture using the commercially available microwell culture system.

Generación de organoides testiculares porcinos con arquitectura específica de Testis utilizando Microwell Culture

Organoids are three dimensional structures composed of multiple cell types that are capable of recapitulating tissue architecture and functions of organs ...