EQUIPO <ul><li> De cultivo de tejidos incubadora a 37 º C, 5% de CO 2 </li><li> Centrífugo </li><li> Juego de micropipetas (100, 100μL) </li><li> Baño de agua 37 ° C </li><li> Baño de agua a 90 ° C para ser fuente de vapor de agua </li><li> Microscopio de contraste de fase </li></ul> PRIMER PASO Tratar las células con colcemid En este procedimiento se utilizó la línea de células madre cultivadas H9 en fibroblastos embrionarios de ratón (MEF) inactivados con mitomicina C (Sigma) y se mantuvieron en DMEM/F12 (Invitrogen), complementado con el reemplazo de octavos de final 20% de suero (KSR, Gibco) y de 8ng/mL factor de crecimiento fibroblástico (FGF-2, R &amp; D). Con el fin de obtener un gran número de los diferenciales de metafases, se recomienda el uso de cultivos con alto índice mitótico en el que hay muchas células que se dividen. <ol><li> Tome el frasco de cultivo de la incubadora y, en el flujo laminar, añadir Colcemid a una concentración final de 0,1 mg / ml. </li><li> Volver el frasco a la incubadora y esperar 3 horas para que el inhibidor puede causar un paro mitótico en metafase, cuando los cromosomas están bien condensada y se pueden visualizar fácilmente bajo el microscopio. </li></ol> La fijación de las células <ol start="3"><li> Saque el medio y agregar tripsina / EDTA 0,05% suficiente para cubrir la superficie de las células. Permitir que las células se incuban durante 5 minutos a temperatura ambiente y luego inactivar la enzima, ya sea por la adición de medio de cultivo que contiene suero o con sólo añadir suero de las células. NOTA: Es importante asegurarse de que una única suspensión de células se realiza para obtener una buena propaga metafase. </li><li> Transferencia de las células a un tubo de centrífuga de 15 ml y centrifugar a 122 g durante 5 minutos. </li><li> Descartar el sobrenadante, dejando alrededor de 200 ml, y resuspender el precipitado suavemente tocando el tubo, como se muestra en el video. Asegúrese de que las células están bien disociado, por lo que no se puede visualizar cualquier grupo en la solución. </li><li> A continuación, tendrá que añadir 5 ml de solución hipotónica (KCl 75 mm) pre-calentado a 37 ° C, lentamente, por la pared del tubo como en la siguiente descripción. Primero se debe agregar 3 ml, invertir el tubo a una posición horizontal justo para mezclar las células con la solución hipotónica a continuación, añadir el resto de 2 ml y mantener caliente a 37 ° C durante 15 minutos. NOTA: En este punto la solución hipotónica se producirá un aumento en el volumen celular y ayudan a desenredar los cromosomas. El tiempo de incubación es importante para adquirir una buena extiende cromosoma. Si el tiempo es demasiado tiempo, la membrana celular puede explotar muy pronto y los cromosomas se han perdido. Si es demasiado corto, puede ser difícil de obtener porque se extiende el cromosoma de la membrana celular no puede alterar. </li><li> Añadir 3 gotas de la solución fijadora (metanol / ácido acético glacial 3:1), invertir el tubo suavemente para mezclar la solución de fijación con las células y se centrifuga a 122 g durante 5 minutos sin interrupción. NOTA: La solución de fijación debe estar recién hecho. NOTA: Es importante la utilización de la centrifugación sin descanso para evitar el desgaste células, evitando la pérdida del material. </li><li> Descartar el sobrenadante, dejando alrededor de 200 ml, y resuspender el precipitado suavemente tocando el fondo del tubo. NOTA: Una vez más, asegúrese de que las células están bien disociado, por lo que no se puede visualizar cualquier grupo en la solución. </li><li> Añadir los primeros 3 ml de solución fijadora, lentamente por la pared del tubo, mientras se gira el tubo. Invierta el tubo a una posición horizontal justo para mezclar las células, y luego añadir más 2 ml de la solución de fijación a la pared del tubo para tomar las células hacia abajo. De archivo a 4 ° C durante la noche. NOTA: Las células fijadas se pueden almacenar en la solución fijadora durante meses a 4 ° C. Además de proteger las células en su estado hinchado, la solución fijadora elimina los lípidos y las proteínas se desnaturaliza. Estos hechos hacen que la frágil membrana de la célula y, en consecuencia, que el cromosoma propaga fácilmente. </li></ol> LIMPIEZA DE LA portaobjetos de vidrio Antes de iniciar la toma se extiende a su cromosoma asegurarse de que las diapositivas se limpian correctamente o es posible que pierda algunos núcleos y también metafases. Este paso es también importante si quieres probar alguna técnica de hibridación 9. <ol><li> Coloque los portaobjetos en un vaso que contiene HCl 6M durante al menos 3 horas a temperatura ambiente. </li><li> Después de este tiempo, lavar los portaobjetos con agua corriente durante 10 minutos. </li><li> Enjuague los portaobjetos en agua destilada y dejar que se sequen a temperatura ambiente. </li><li> Ahora las diapositivas se pueden almacenar en 96% de etanol y se seca con un paño inmediatamente antes del uso. </li></ol> NOTA: Para los métodos de hibridación in situ, las diapositivas no se pueden almacenar durante más de 2 días en etanol al 96% SEGUNDO PASO Lavado de las células <ol start="10"><li> Al día siguiente, centrifugar las células a 122 g durante 5 minutos sin interrupción. </li><li> Discard el sobrenadante dejando alrededor de 200 ml. Resuspender el precipitado agitando suavemente el tubo a continuación, añadir 5 ml de solución fijadora fresca (metanol / ácido acético glacial 3:1), lentamente por la pared del tubo mientras se gira el tubo. Repita este paso de lavado dos veces más para un total de tres lavados. NOTA: La solución de fijación debe estar recién hecho. </li></ol> Preparación de las diapositivas <ol start="12"><li> En la última etapa de centrifugación dejar una cantidad suficiente de solución para homogeneizar el sedimento celular y obtener una densidad adecuada de las células en las diapositivas (ver figura 1). </li><li> Añadir 20-30 l de suspensión de células en un portaobjetos limpio y seco, moviendo la pipeta muy cerca y en paralelo a la superficie. Medida que se evapora la solución de fijación, la superficie de deslizamiento se convierte en granulado. </li><li> Inmediatamente, exponer la diapositiva, boca arriba, en el vapor de agua caliente (90 °) durante 30 segundos para hacer que las células explotan. NOTA: En este momento, el metanol se evapora de la solución de fijación, lo que resulta en una mayor concentración de ácido acético, que junto con el agua estimula la difusión de los cromosomas. </li><li> Mira a un microscopio de contraste de fase sólo para comprobar si la concentración, así como la distribución de las células es buena (ver figura 1). </li><li> Ahora las diapositivas están listos para ser utilizados para comprobar el juego de cromosomas mediante enfoques como la citogenética de bandas G (Giemsa), FISH, SKY y CGH. </li></ol> CONSEJOS IMPORTANTES ¿Cómo elegir una metafase adecuados para ser analizados La elección de metafases redondas y aisladas (figura 2D), es posible evitar la consideración de aneuploidías falsa como la ganancia de los cromosomas generados por dos o más se extiende mezclados entre sí o la pérdida de cromosomas generados por un cromosoma en marcha. Por lo tanto, evitar la elección de metafases cerca de los núcleos, ya que algunos cromosomas pueden estar ocultos por ellos (Figura 2A y 2B). Además, busca metafases ronda y estar seguros de que cualquier cromosoma se separa de la metafase (figura 2C). Células alimentadoras y el cariotipo células madre De bandas G y análisis SKY, la presencia de células alimentadoras en la cultura células madre no interferir con los resultados, porque las células de alimentación no están bajo un estado de división (inactivada por la mitomicina C o irradiación) y no será parte de la población de la metafase. Sin embargo, para la aplicación de la técnica de FISH en núcleos en interfase, es preferible separar a la población de células madre a las células de alimentación por la recolección de las colonias manualmente antes de tripsinización para el análisis de FISH. <img border="0" src="/files/ftp_upload/1512/1512fig1tmb.jpg" alt="Figura 1" /> Figura 1: ¿Cómo saber la densidad celular apropiada en las diapositivas. Las imágenes de microscopio de contraste de fase en el objetivo de 10x. (A) La densidad celular es muy alta. Las flechas indican la metafase se propaga muy cerca de los núcleos. (B) Una diapositiva con una buena densidad de las células. Los círculos muestran los diferenciales metafases aislado de núcleos o metafases otros. Por favor, <a href="https://www.jove.com/files/ftp_upload/1512/1512fig1.jpg">haga clic aquí</a> para ver una versión ampliada de la figura 1. <img border="0" src="/files/ftp_upload/1512/1512fig2tmb.jpg" alt="Figura 2" /> Figura 2: ¿Cómo elegir una metafase adecuados para ser analizados. Los cromosomas son teñidos con DAPI y las imágenes obtenidas por un microscopio de fluorescencia en el objetivo de 100x. (A) (B) Evitar metafases cerca de los núcleos (indicado por las flechas) (C) y no metafases ronda que los cromosomas presentan separados (círculo). (D) A la metafase ronda es una metafase bueno para ser analizados. Por favor, <a href="https://www.jove.com/files/ftp_upload/1512/1512fig2.jpg">haga clic aquí</a> para ver una versión ampliada de la figura 2.

<ol>
	<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 (5391), 1145-1145 (1998).</li><li>Draper, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+gain+of+chromosomes+17q+and+12+in+cultured+human+embryonic+stem+cells.">Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells.</a> Nat Biotechnol. 22 (1), 53-53 (2004).</li><li>Maitra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+alterations+in+cultured+human+embryonic+stem+cells.">Genomic alterations in cultured human embryonic stem cells.</a> Nat Genet. 37 (10), 1099-1099 (2005).</li><li>Mitalipova, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preserving+the+genetic+integrity+of+human+embryonic+stem+cells.">Preserving the genetic integrity of human embryonic stem cells.</a> Nat Biotechnol. 23 (1), 19-19 (2005).</li><li>Buzzard, J. J., Gough, N. M., Crook, J. M., Colman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Karyotype+of+human+ES+cells+during+extended+culture.">Karyotype of human ES cells during extended culture.</a> Nat Biotechnol. 22 (4), 381-381 (2004).</li><li>Caisander, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+integrity+maintained+in+five+human+embryonic+stem+cell+lines+after+prolonged+in+vitro+culture.">Chromosomal integrity maintained in five human embryonic stem cell lines after prolonged in vitro culture.</a> Chromosome Res. 14 (2), 131-131 (2006).</li><li>Imreh, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+culture+conditions+favoring+selection+of+chromosomal+abnormalities+in+human+ES+cells.">In vitro culture conditions favoring selection of chromosomal abnormalities in human ES cells.</a> J Cell Biochem. 99 (2), 508-508 (2006).</li><li>Robin-Wesselschmidt, J. L., Loring, J. F., Robin-Wesselschmidt, J. L., Robin-Wesselschmidt, S. c. h. w. a. r. t. z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Stem Cell Manual. , 59-59 (2007).</li><li>Rooney, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Cytogenetics. , (2001).</li><li>Heslop-Harrison, P., Schwarzacher, T., Heslop-Harrison, P., Schwarzacher, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Practical in situ hybridization. , 51-51 (2000).</li></ol>

The authors have nothing to disclose.

La preparación de los diferenciales de los cromosomas es un paso crítico para el éxito de un análisis de la situación genética de las células madre embrionarias mediante técnicas de rutina como el G-bandas, y las técnicas más sofisticadas como el pescado, SKY y CGH. Este procedimiento se puede aplicar a muchos tipos de células, variando el período de incubación colcemid, que depende de la duración del ciclo celular. Para las colonias de células madre embrionarias, que esperar ...

<table><tbody><tr><td>15 ml centrifuge tube</td><td>Techno Plastic Products</td><td>91015</td><td></td></tr><tr><td>Colcemid Karyo MAX</td><td>GIBCO, by Life Technologies</td><td>15212-012</td><td></td></tr><tr><td>EDTA</td><td>Isofar</td><td>721</td><td></td></tr><tr><td>Glacial acetic acid</td><td>Isofar</td><td>100</td><td></td></tr><tr><td>Methanol</td><td>Isofar</td><td>208</td><td></td></tr><tr><td>Potassium Chloride (KCl)</td><td>Merck &amp;amp; Co., Inc.</td><td>104.931.000</td><td></td></tr><tr><td>Slide</td><td>Bioslide</td><td>7105-1</td><td></td></tr><tr><td>Tripsin</td><td>Sigma-Aldrich</td><td>T4799</td><td></td></tr></tbody></table>

chromosomal spread preparation of human embryonic stem cells for karyotyping

Aunque las células madre embrionarias (células madre) se ha demostrado que presentan un cariotipo diploide estable 1, muchos estudios han reportado que en función de las condiciones de cultivo se convierten propensos a adquirir las anomalías cromosómicas como la suma de la totalidad o partes de los cromosomas. De hecho, durante el cultivo prolongado, alteraciones del cariotipo se observan cuando la disociación enzimática o química se utilizan 2,3,4, mientras que la disección manual de las colonias de pases conserva un cariotipo estable 5. Además, los cambios en el medio ambiente tales como la eliminación de las células de alimentación también parecen poner en peligro la integridad genética de las células madre a 3,6. Una vez que las alteraciones cromosómicas pueden afectar la fisiología celular, la caracterización de la integridad genética de las células madre in vitro células madre es esencial considerar como una herramienta esencial en los estudios de la embriogénesis y la prueba de la droga. Por otra parte, para el futuro con fines terapéuticos cambios cromosómicos son una preocupación real, ya que se asocia frecuentemente a la carcinogénesis. Aquí mostramos un método sencillo y útil para obtener alta calidad se extiende cromosoma para su posterior análisis del cromosoma establecidos por bandas G, FISH, SKY o técnicas de CGH 7,8. Se recomienda comprobar el estado cromosómico de rutina, con intervalos de 5 pasajes a fin de vigilar la aparición de las translocaciones y aneuploidías Priscila Britto y Sartore Rafaela contribuido igualmente a la de papel.

Watch this Scientific Journal Video about Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping at JoVE.com

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

El cariotipo es una técnica sencilla y útil ampliamente utilizado para la detección de alteraciones genéticas. A continuación se describe paso a paso el protocolo para la preparación de los cromosomas propagación de células madre embrionarias humanas para controlar el estado cromosómico de estas células se mantienen en cultivo.

Preparación Extienda los cromosomas de las células estaminales embrionarias para cariotipo

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

chromosomal-spread-preparation-human-embryonic-stem-cells-for

Research

JoVE Journal

Biology

48.6K vistas.  Federal University of Rio De Janeiro-UFRJ. El cariotipo es una técnica sencilla y útil ampliamente utilizado para la detección de alteraciones genéticas. A continuación se describe paso a paso el protocolo para la preparación de los cromosomas propagación de células madre embrionarias humanas para controlar el estado cromosómico de estas células se mantienen en cultivo.

Video: Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

Generación de células madre pluripotentes inducidas a partir de Frozen Buffy Coats utilizando para no integrar episomal plásmidos

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Investigación

Biología del Desarrollo

Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries. In comparison, prophase spermatocytes can be collected from juvenile male mice with higher yields without the need for microdissection. However, it is difficult to obtain a pure synchronized population of cells at specific stages due to the heterogeneity of meiotic and post-meiotic germ cell populations in the juvenile and adult testis. For later stages of meiosis, it is advantageous to assess oocytes undergoing meiosis I (MI) or meiosis II (MII), because groups of mature oocytes can be collected from adult female mice and stimulated to resume meiosis in culture. Here, methods for meiotic chromatin spread preparations using oocytes dissected from fetal, neonatal and adult ovaries are described with accompanying video demonstrations. Chromosome missegregation events in mammalian oocytes are frequent, particularly during MI. These techniques can be used to assess and characterize the effects of different mutations or environmental exposures during various stages of oogenesis. As there are distinct differences between oogenesis and spermatogenesis, the techniques described within are invaluable to increase our understanding of mammalian oogenesis and the sexually dimorphic features of chromosome and protein dynamics during meiosis.

Oogenesis in mammals is known to be error-prone, particularly due to chromosome missegregation. This manuscript describes chromatin spread preparation methods for mouse prophase, metaphase I and II-staged oocytes. These fundamental techniques allow for the study of chromatin-bound proteins and chromosome morphology throughout mammalian oogenesis.

Cromatina difundir preparados para el análisis de progresión de ovocitos de ratón de profase a metafase II

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for ...

Genética

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell&#39;s endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent&#160;stem cells.

Genome editing of human pluripotent stem cells (hPSCs) can be done quickly and efficiently. Presented here is a robust experimental procedure to genetically engineer hPSCs as exemplified by editing the AAVS1 safe harbor locus to express EGFP and introduce antibiotic resistance.

Establecimiento de editados-Genoma Humano pluripotentes Líneas de Células Madre: De Focalización de Aislamiento

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human ...

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

Cariotipo espectral para el Estudio de anomalías cromosómicas en humanos y ratones con la enfermedad renal poliquística

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

Medicina

Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to be responsible for carcinogenesis in such tissues. Although proliferative polyploid cells are requisite for analyzing chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is rather challenging. Induction of tetraploidy by chemical agents usually results in a mixture of diploid and tetraploid populations, and most studies employed fluorescence-activated cell sorting or cloning by limiting dilution to separate tetraploid from diploid cells. However, these procedures are time-consuming and laborious. The present report describes a relatively simple protocol to induce proliferative tetraploid cells from normal human fibroblasts with minimum contamination by diploid cells. Briefly, the protocol is comprised of the following steps: arresting cells in mitosis by demecolcine (DC), collecting mitotic cells after shaking off, incubating collected cells with DC for an additional 3 days, and incubating cells in drug-free medium (They resume proliferation as tetraploid cells within several days). Depending on cell type, the collection of mitotic cells by shaking off might be omitted. This protocol provides a simple and feasible method to establish proliferative tetraploid cells from normal human fibroblasts. Tetraploid cells established by this method could be a useful model for studying chromosome instability and the oncogenic potential of polyploid human cells.

Although proliferative polyploid cells are necessary to analyze chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is not easy. The present report describes relatively simple procedures to establish proliferative tetraploid cells free of a diploid population from normal human fibroblasts.

Establecimiento de células tetraploides proliferativa de fibroblastos humanos no transformadas

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

Inmunoprecipitación de la cromatina de células estaminales embrionarias humanas

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Biología

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Adultas y embrionarias del músculo esquelético Cultura Microexplant y aislamiento de células madre de músculo esquelético

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome&#160;and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births1,2, 60-80% of all miscarriages3,4, 10% of stillbirths2,5, 13% of individuals with congenital heart disease6, 3-6% of infertility cases2, and in many patients with developmental delay&#160;and birth defects7. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance8,9.&#160; Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents10-13.
Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy&#39;s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15.

Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY).

Cromosoma Preparación de las células cultivadas

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell populations.&#160;These cultures contain the cells of interest, but are also contaminated with undifferentiated ESCs, non-neural derivatives and other neuronal subtypes.&#160; This limits their use in in vitro and in vivo applications, such as in vitro modeling for drug discovery or cell replacement therapy. To help overcome this, reporter cell lines, which offer a means to visualize, track and isolate cells of interest, can be engineered.&#160;However, to achieve this in human embryonic stem cells via conventional homologous recombination is extremely inefficient.&#160;This protocol describes targeting of the Pituitary homeobox 3 (PITX3) locus in human embryonic stem cells using custom designed zinc-finger nucleases, which introduce site-specific double-strand DNA breaks, together with a PITX3-EGFP-specific DNA donor vector. Following the generation of the PITX3 reporter cell line, it can then be differentiated using published protocols for use in studies such as in vitro Parkinson&#8217;s disease modeling or cell replacement therapy.

Reporter cell lines offer a means to visualize, track and isolate cells of interest from heterogeneous populations.&#160;However, gene-targeting using conventional homologous recombination in human embryonic stem cells is extremely inefficient. Herein, we describe targeting CNS midbrain specific transcription factor PITX3 locus with EGFP using zinc-finger nuclease enhanced homologous recombination.

Nucleasa dedo de zinc-gen mejorada en células madre embrionarias humanas

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell ...

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on the surface of slides (rather than dropping them from a height), we demonstrate an optimized technique on mouse spermatocytes that was first described in 1997. This method is widely used in laboratories to study mammalian meiosis because it yields a plethora of high quality nuclei undergoing substages of prophase I. Seminiferous tubules are first placed in a hypotonic solution to swell spermatocytes. Then spermatocytes are released into a sucrose solution to create a cell suspension, and nuclei are spread onto fixative-soaked glass slides. Following immunostaining, a diversity of proteins germane to meiotic processes can be examined. For example, proteins of the synaptonemal complex, a tripartite structure that connects the chromosome axes/cores of homologs together can be easily visualized. Meiotic recombination proteins, which are involved in repair of DNA double-strand breaks by homologous recombination, can also be immunostained to evaluate progression of prophase I. Here we describe and demonstrate in detail a technique widely used to study mammalian meiosis in spermatocytes from juvenile or adult male mice.

Meiosis is the developmental process by which gametes are formed through a single round of DNA replication and two successive rounds of chromosome segregation. Mammalian meiosis can be examined by utilizing a technique to prepare meiotic chromosome spreads. Here, we demonstrate a method of preparing surface-spread nuclei from mouse spermatocytes.

Preparación del cromosoma meiótico se extiende desde ratón espermatocitos

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on ...