Gli autori ringraziano Brian Muntean, Shao Lo, Maki Takahashi e Blair Mell per la loro assistenza tecnica. Questo lavoro è stato sostenuto da premi dal NIH (DK080640) e The University of Translational Research Award stimolazione Toledo &amp; Promedica al dottor Surya Nauli.

1. Cella pretrattamento e la preparazione metafase <ol><li> Le cellule sono cresciute in modif Dulbeccòs di Medio di Eagle (DMEM) contenente 10-15% siero bovino fetale (FBS) e 1% penicillina / streptomicina a 37 ° C con 5% CO 2 incubatore, fino a raggiungere 70-80% confluenza. </li><li> Trattare le cellule con soluzione Colcemid a 0,05 ug / ml per 30-60 min. </li><li> Raccogliere il terreno contenente le cellule galleggianti da 50 ml sterili provette Falcon. Lavare le cellule sulla piastra con sterile1X-PBS. Dopo aver incubato con tripsina sterile per 1-2 min, raccolto e raccogliere le cellule rimanenti nel tubo stesso. </li><li> Centrifugare le provette a 1000 rpm per 5 min, aspirare il surnatante lasciando 0,5 ml e allentare pellet da sfogliando con un dito solo. </li><li> A seconda delle dimensioni pellet, aggiungere 5-10 ml di soluzione ipotonica dello 0,56% KCl in dH 2 O e incubare la sospensione a 37 ° C per 30-45 min. </li><li> Aggiungere una goccia di metanolo / acido acetico(3:1 vol / vol) per ml di sospensione cellulare ipotonico e invertire delicatamente il tubo per la miscelazione. </li><li> Centrifugare a 1200 rpm per 5 minuti e raccogliere il pellet come step 1.4, quindi aggiungere 5 ml di metanolo dolce / acido acetico (3:1 vol / vol) goccia a goccia la soluzione fissativa, mentre sfogliando il pellet continuamente. Questa procedura è critica così che i metafase non verrà intrappolato nella ciuffi delle cellule &#39;che comprometterebbero l&#39;esperimento. </li><li> Centrifugare nuovamente a 1200 rpm per 5 min e aggiungere 5-10 ml di ghiaccio-freddo metanolo / fissativo acido acetico lungo la parete del tubo. In questa fase, se necessario, le cellule possono essere conservate in soluzione fissativa in un tubo stretto e sigillato a -20 ° C per breve termine o -80 ° C per lungo termine (anni) per uso futuro. </li><li> Vetrini Pulizia in etanolo assoluto, quindi immergerlo in dH 2 O per approximately10X per formare una guaina di acqua sulla superficie del vetrino. Porre il vetrino su una piastra di vetro e calo del 15-20μl di sospensione cellulare (dal punto 1,8) Da 10 &quot;sopra il vetrino. Porre il vetrino in un bagnomaria regolato a 65-70 ° C per 1-2 minuti e lasciare asciugare. </li><li> Controllare il vetrino sotto un microscopio ottico con obiettivi a secco 10X e 40X assicurandosi che vi sia cromosomi in metafase e gli spread sono distribuiti uniformemente. Verifica della presenza citoplasma che circonda i cromosomi. Se citoplasma è presente procedere con pretrattamento diapositiva (digestione pepsina), se non citoplasma è presente e le cromosomi hanno buona morfologia, allora non c&#39;è bisogno di pretrattamento diapositiva. </li></ol> 2. Presentazione pretrattamento (pepsina trattamento) <ol><li> Applicare 120 ml di soluzione 1:200 RNasi (20 mg / ml) disciolto in 2X-SSC su un 24 mm coverglass microscopio x 60 mm e invertire la metafase faccia scivolare sul coverglass poi capovolgere delicatamente la faccia slitta metafase e incubare a 37 ° C per 45 min. </li><li> Rimuovere con cautela il vetrino coprioggetti senza graffiare il vetrino e lavare in SSC 2X-buffer in una vaschetta Coplin ogni 5 min per 15 mincon agitazione. </li><li> Aggiungere 5-15 pl di soluzione di archivio di pepsina (100 mg / ml in dH 2 O) in un bicchiere pulito e quindi aggiungere 100 ml di preriscaldato (37 ° C) 0.01M HCl. È importante che la pepsina viene aggiunto in un pulito becher prima e non direttamente nella soluzione di HCl, altrimenti non sarebbe sciogliere in soluzione. Incubare il vetrino in una vaschetta Coplin, contenente la HCl / pepsina soluzione a 37 ° C per 3-5 min. Questo passaggio è molto critica come digestione troppo causerà i cromosomi da overdigested e troppo digestione poco lascerà il citoplasma non digerito che potrebbe portare ad legame non specifico della sonda e interferire con il segnale di ibridazione. </li><li> Lavare il vetrino in una vaschetta Coplin contenente 100 ml 1X-PBS per 5 min due volte a temperatura ambiente. </li><li> Lavare il vetrino in una vaschetta Coplin contenente 100 ml 1X-PBS/MgCl 2 per 5 min a temperatura ambiente (50 ml di 1M MgCl 2 in 950 ml di 1X-PBS). </li><li> Mettere il vetrino in una vaschetta Coplin containing 100 ml di formaldeide 1% per 10 min a temperatura ambiente (1,7 ml del 37% formaldeide in 100 ml di 1X-PBS/MgCl 2). </li><li> Lavare il vetrino in una vaschetta Coplin contenente 100 ml 1X-PBS per 5 min. </li><li> Osservare il vetrino sotto un microscopio ottico 40X con lenti a secco per assicurare che i vetrini vengono digeriti correttamente e non citoplasma è presente e la morfologia dei cromosomi è conservato. Selezionare un&#39;area per l&#39;ibridazione con una penna di diamante. </li></ol> 3. Cromosoma e sonda denaturazione e l&#39;ibridazione <ol><li> Preparare la soluzione fresca denaturazione (70% formamide/2X SSC, pH 7.0) e preriscaldare a 70-80 ° C in una vaschetta Coplin posto in un luogo bagnomaria il vetrino nel vaso Coplin contenente la soluzione di denaturazione a bagnomaria a 70 ° C per i cromosomi del mouse e 80 ° C per i cromosomi umani per anni &#39;30-1.5 min. </li><li> Disporre il vetrino in ghiacciato etanolo al 70% per 3 min seguita da 80% e 100% di etanolo per 3 minuti ciascuna e lasciare asciugare all&#39;aria. Esaminare il vetrinoper morfologia cromosoma. Buona la morfologia dei cromosomi si indica con cromosomi scure e non &quot;phase-luce&quot; o halo cromosomi. </li><li> Riscaldare la sonda SkyPaint (vernice kit SKY; flaconcino # 1) a 37 ° C con agitazione per 20 min, vortex e centrifugare brevemente a 1000 rpm per pochi secondi. </li><li> Denaturare il sonda in un termociclatore programmato per un due-step ciclo a 85 ° C per 5 ciclo min seguita da 37 ° C per 60 min per consentire marcato-sonda di DNA per preannealing. </li><li> Applicare 10 pl della sonda denaturata sull&#39;area di ibridazione e coprire con un mm 22 x 40 millimetri coprioggetti facendo attenzione a non intrappolare bolle d&#39;aria. Sigillare i bordi del coverglass con cemento gomma e incubare in una camera umidificata a 37 ° C per 48-72 ore. </li></ol> 4. Sonda di rilevamento della fluorescenza <ol><li> Rimuovere il copri con cura e porre il vetrino in una vaschetta Coplin contenente preriscaldato (45 ° C) la soluzione di lavaggio I (preparata formamide 50% in 2X SSC). Lavare per 5 mintre volte a 45 ° C in un bagno d&#39;acqua agitando a 45 rpm </li><li> Lavare il vetrino nella soluzione di lavaggio II (1X SSC) a 45 ° C per 5 min due volte con agitazione. </li><li> Lavare il vetrino nella soluzione di lavaggio III (4X SSC/0.1% Tween 20) per 5 min a 45 ° C con agitazione. </li><li> Applicare 80 microlitri di reagente bloccante (paint kit SKY; flacone # 2), inserire un coprioggetto e incubare a 37 ° C per 30 min. </li><li> Rimuovere il vetrino e consentire al fluido di drenare. Applicare 80 microlitri di reagente di colorazione Cy5 (Concentrated Ricerca anticorpi CAD kit; flacone # 3), applicare il coprioggetti e incubare a 37 ° C per 40 min. </li><li> Lavare il vetrino con soluzione di lavaggio III a 45 ° C per 5 min tre volte con agitazione. </li><li> Applicare 80 microlitri di reagente Cy5.5 colorazione (Concentrated Ricerca anticorpi CAD kit; fiala # 4), posizionare il coprioggetti e incubare a 37 ° C per 40 min. </li><li> Lavare il vetrino con soluzione di lavaggio III a 45 ° C per 5 min tre volte con agitazione. </li><li> Inclinare la diapositiva e consentire il fluido<html> scolare. Applicare 20 l di anti-fade reattivo DAPI (paint kit SKY; flaconcino # 5) e mettere da 24 mm coverglass microscopio x 60 mm. Rimuovere con cura le bolle d&#39;aria che potrebbero essere formate. Le diapositive possono essere ripreso immediatamente oppure conservati a 4 ° C al buio per non più di 1 settimana. </li></ol> 5. Acquisizione di immagini e analisi <ol><li> Acquisizione Immagine è compiuta da visualizzando vetrini metafase usando un microscopio Olympus equipaggiata con un obiettivo 60X immersione in olio, un cubo Spectral (progettato su misura tripla banda filtro passa), un filtro DAPI e un modulo interferometro Sagnac con una telecamera CCD. </li><li> Spettrale cariotipi sono state effettuate utilizzando il software View SKY (Applied Version immagini spettrali 1,62) seguendo il manuale dell&#39;utente. </li><li> Dopo aver analizzato le immagini, i cromosomi possono essere visti come immagini a colori (con appositi colori fluorescenti), immagini a colori pseudo (con i colori per la classificazione) e invertiti immagini DAPI (specifico disegno a strisce). </li></ol>e_title &quot;&gt; 6. rappresentativi Risultati Una procedura di SKY completa dura solitamente circa una volta alla settimana (Figura 1). Ciò include l&#39;acquisizione delle immagini e l&#39;analisi a condizione che le cellule in metafase sono in quantità adeguata. Analisi Karyotyping rivela cariotipo normale topo (40, XY) di cellule provenienti da topi wild type (Figura 2a). In contrasto, cellule da PKD1 - / - topo (modello topo PKD) mostra un significativo aumento in numero dei cromosomi e anomalie strutturali, quali delezioni cromosomiche (cromosoma # 8) e le traslocazioni (cromosomi # 11 e 19) (Figura 2b). Abbiamo anche analizzato i tessuti vascolari dai pazienti ADPKD. Uno studio semplice contando i numeri cromosomiche indicato che non-ADPKD e alcuni campioni ADPKD vascolari avuto normali numeri cromosomiche di 23 coppie (Figura 2c). In generale, però, abbiamo osservato fallimento di segregazione cromosomica, con conseguente in 46 coppie di cromosomi in ACampioni DPKD invece di 23 coppie (figura 2d). <img alt="Figura 1" src="/files/ftp_upload/3887/3887fig1.jpg" /> Figura 1. Diagramma di flusso protocollo di SKY. Diagramma di flusso del protocollo di SKY illustra i passaggi per completare un esperimento a partire dal pre-trattamento delle cellule in metafase e preparati per l&#39;acquisizione delle immagini e analisi. Una approssimativa di una settimana timeline è presentato a sinistra con passo-passo le procedure per ogni giorno. <img alt="Figura 2" src="/files/ftp_upload/3887/3887fig2.jpg" /> Figura 2. Cariotipo spettrale del mouse su linee cellulari e immortalati appena isolato le cellule primarie umane. Colori e immagini capovolte DAPI di cromosomi singoli mostrato prima l&#39;ordinamento cromosomico. Dopo la cernita, i cromosomi sono presentati nella tabella &quot;classificazione&quot;. A) l&#39;immagine di SKY uno spread metafase dal topo wild-type contiene cariotipo normale (40, XY). B) </strong&gt; SKY immagine di uno spread metafase da PKD1 - / -. del mouse mostra il numero dei cromosomi anormali (68 invece di 40) e le anomalie cromosomiche come le delezioni cromosomiche (cromosoma # 8) e le traslocazioni cromosomiche (cromosomi # 11 e 19) c) SKY immagine di uno spread metafase dal tessuto vascolare di un non-ADPKD paziente ha cariotipo normale (46, XX). d) immagine SKY di uno spread metafase dal tessuto vascolare di un paziente ADPKD contiene cariotipo anormale (92, XXXX). Parti dei dati sono stati riportati in precedenza e riutilizzato con autorizzazione 14.

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Cariotipo spettrale (SKY) è una tecnica utilizzata nello studio di citogenetica composizioni genomiche e cromosomiche. Questa tecnica si avvale di sonde cromosoma pittura, e il rilevamento di queste sonde sono stati acquisiti tramite un interferometro di Sagnac. Il processo di SKY completo dura di solito circa una settimana, e coinvolge diversi passaggi chiave (Figura 1). La SKY utilizza un protocollo standard, che è stato descritto da Padilla-Nash et al 3. Il protocollo è stato p...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> DMEM </td><td> Cellgro Mediatech Inc. </td><td> 10-013-CV </td><td></td></tr><tr><td> Siero bovino fetale (FBS) </td><td> Hyclone </td><td> SH30088-03 </td><td></td></tr><tr><td> Penecillin / streptomicina </td><td> Thermo Scientific </td><td> SV30010 </td><td></td></tr><tr><td> Colcemid </td><td> Roche Diagnostics </td><td> 10 295 892 001 </td><td> 10 pg / ml </td></tr><tr><td> HCl </td><td> Fisher Scientific </td><td> A144-500 </td><td></td></tr><tr><td> KCl </td><td> Fisher Scientific </td><td> S77375-1 </td><td></td></tr><tr><td> Fosfato soluzione salina tamponata </td><td> Thermo Scientific </td><td> SH30256-01 </td><td></td></tr> &lt;tr&gt; <td> SKY paint kit sonda (Human) </td><td> Applicata Imaging Spectral </td><td> SKY000028 </td><td></td></tr><tr><td> SKY vernice sonda kit (mouse) </td><td> Applicata Imaging Spectral </td><td> SKY000030 </td><td></td></tr><tr><td> Concentrato kit per il rilevamento degli anticorpi </td><td> Applicata Imaging Spectral </td><td> SKY000033 </td><td></td></tr><tr><td> Tripsina </td><td> Thermo Scientific </td><td> SH30236.01 </td><td></td></tr><tr><td> Metanolo </td><td> Fisher Scientific </td><td> A433P-4 </td><td></td></tr><tr><td> Acido acetico </td><td> Fisher Scientific </td><td> A38-212 </td><td></td></tr><tr><td> RNasi A </td><td> Roche Diagnostics </td><td> 10 109 169 001 </td><td></td></tr><tr><td> Pepsina </td><td> Sigma-Aldrich </td><td> P6887-5G </td><td></td></tr><tr><td> MgCl 2 </td><td> Fluka </td><td> 63069-500ML </td><td></td></tr><tr><td> 37% Formaldeide </td><td> Mallinckrodt Baker </td><td> 2106-02 </td><td></td></tr><tr><td> 20X SSC </td><td> Promega </td><td> V4261 </td><td></td></tr><tr><td> Formammide </td><td> Fluka </td><td> 47671 </td><td> Preparare al momento dell&#39;uso </td></tr><tr><td> Tween-20 </td><td> Fisher Scientific </td><td> BP337-500 </td><td></td></tr><tr><td> Microscopio diapositive di vetro </td><td> Fisher Scientific </td><td> 12-549 </td><td></td></tr><tr><td> Microscopio copertura in vetro 24x60mm </td><td> VWR Vista Vision </td><td> 16004-312 </td><td></td></tr><tr><td> Mastice </td><td> Elmer (locali negozio al dettaglio) </td><td></td><td></td></tr><tr><td> Ibridizzazione / humidifiedchamber / Tray </td><td> Simport </td><td> M920-2 </td><td> mettere gli asciugamani di carta bagnati in fondo </td></tr><tr><td> Thermocycler </td><td> Eppendorf </td><td> Epgradient S </td><td></td></tr><tr><td> Scuotendo la piattaforma / Agitatore orbitale </td><td> Bellco Biotecnologie </td><td></td><td></td></tr><tr><td> Scuotendo bagno / acqua </td><td> Rigore scientifico </td><td></td><td></td></tr><tr><td> DAPI filtro cubo </td><td> Tecnologia Chroma </td><td></td><td></td></tr><tr><td> SKY filtro cubo </td><td> Tecnologia Chroma </td><td></td><td></td></tr><tr><td> SpectraCube </td><td> Applicata Imaging Spectral </td><td></td><td></td></tr><tr><td> Inverted cella microscopio cultura </td><td> Nikon </td><td> Nikon Eclipse TS100 </td><td></td></tr><tr><td> Microscopio a fluorescenza </td><td> Olimpo </td><td> IX70 </td><td> Olio 60X </td></tr></tbody></table>

spectral karyotyping to study chromosome abnormalities in humans and mice with polycystic kidney disease

Metodo convenzionale per identificare e classificare i singoli cromosomi dipende dal modello unico bande di ciascun cromosoma in una determinata specie in fase di analisi 1, 2. Questa tecnica classica banding, tuttavia, non è affidabile nell&#39;identificare complesse aberrazioni cromosomiche come quelle associate con il cancro. Per superare i limiti della tecnica banding, cariotipo spettrale (SKY) viene introdotto per fornire molte informazioni affidabili sulle anomalie cromosomiche. SKY è una fluorescenza multicolor ibridazione in situ (FISH), tecnica per rilevare cromosomi in metafase, con microscopio spettrale 3, 4. SKY ha dimostrato di essere uno strumento prezioso per l&#39;analisi citogenetica di una vasta gamma di anomalie cromosomiche associate con un gran numero di malattie genetiche e tumori maligni 5, 6. SKY implica l&#39;uso di sonde di DNA multicolor marcato in fluorescenza preparati dal degenerato oligonucleprimers otide mediante PCR. Così, ogni cromosoma ha un colore univoco spettrale dopo in-situ ibridazione con sonde, che sono differenzialmente etichettati con una miscela di coloranti fluorescenti (Rodamina, Texas Red, Cy5, FITC e Cy5.5). Le sonde utilizzate per SKY contenere fino a 55 sonde cromosoma specifiche 7-10. La procedura per SKY prevede diversi passaggi (Figura 1). SKY richiede la disponibilità di cellule con elevato indice mitotico da tessuto normale o malato o di sangue. I cromosomi di una cellula singola da o una cella isolate di fresco primaria o una linea cellulare sono diffuse su un vetrino. Questo spread cromosoma è etichettato con una diversa combinazione di coloranti fluorescenti specifici per ciascun cromosoma. Per il rilevamento sonda e acquisizione delle immagini, il sistema di imaging spettrale consiste di interferometro Sagnac e una telecamera CCD. Questo permette misurazione dello spettro di luce visibile emessa dal campione e di acquisire una immagine spettrale frocromosomi m individuali. HiSKY, il software utilizzato per analizzare i risultati delle immagini catturate, fornisce una facile identificazione di anomalie cromosomiche. Il risultato finale è uno metafase e una immagine classificazione cariotipo, in cui ciascuna coppia di cromosomi ha un colore distinto (Figura 2). Questo permette una facile identificazione delle identità e le traslocazioni cromosomiche. Per ulteriori informazioni, visitare il sito Applied Spectral Imaging ( <a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a> ). SKY è stato recentemente utilizzato per l&#39;identificazione di difetti segregazione dei cromosomi e le anomalie cromosomiche negli esseri umani e nei topi con malattia autosomica dominante del rene policistico (ADPKD), una malattia genetica caratterizzata da una disfunzione in cilia primaria 11-13. Utilizzando questa tecnica, abbiamo dimostrato la presenza di segregazione dei cromosomi anormali e difetti cromosomici in pazienti ADPKD e modelli di topo 14. Ulteriori analisi basate SKY non solo ci ha permesso di identificare il numero cromosomico e identità, ma anche per rilevare con precisione le aberrazioni cromosomiche molto complessi come le delezioni e le traslocazioni cromosomiche (Figura 2).

Watch this Scientific Journal Video about Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease at JoVE.com

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

Cariotipo spettrale (SKY) è una tecnica avanzata citogenetica per individuare aberrazioni genomiche e cromosomiche. Questa tecnica si avvale di sonde cromosomiche pittura, che permettono la classificazione di tutti i cromosomi. SKY può anche identificare aberrazioni cromosomiche complesse e difetti segregazione nei topi e nell&#39;uomo con varie patologie, tra cui la malattia del rene policistico.

Cariotipo spettrale allo studio anomalie cromosomiche negli esseri umani e nei topi con malattia del rene policistico

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

spectral-karyotyping-to-study-chromosome-abnormalities-humans-mice

Research

JoVE Journal

Medicine

38.3K Views.  University of Toledo, College of Pharmacy and Pharmaceutical Sciences. Cariotipo spettrale (SKY) è una tecnica avanzata citogenetica per individuare aberrazioni genomiche e cromosomiche. Questa tecnica si avvale di sonde cromosomiche pittura, che permettono la classificazione di tutti i cromosomi. SKY può anche identificare aberrazioni cromosomiche complesse e difetti segregazione nei topi e nell'uomo con varie patologie, tra cui la malattia del rene policistico.

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

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

Rilevazione di Inter-aberrazioni cromosomiche stabili per fluorescenza multipla<em&gt; In Situ</em&gt; Ibridazione (mFISH) e spettrale cariotipo (SKY) in topi irradiati

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

Ricerca

Genetica

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

Usando i Oocytes del Mouse per valutare la funzione del Gene umano durante la meiosi I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

Analisi rapida di aberrazioni cromosomiche in linfociti B mouse da PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Immunologia e infezioni

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

L&#39;uso di ultra-alto campo MRI in piccolo roditore modelli di malattia del rene policistico per<em&gt; In Vivo</em&gt; Fenotipizzazione and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Medicina

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

Chromosomics: Rilevamento delle alterazioni numeriche e strutturali in tutti i 24 cromosomi umani simultaneamente utilizzando un nuovo metodo di analisi FISH OctoChrome

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

Biologia

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 Preparazione da cellule in coltura

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

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish5. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria &#8216;dipstick&#8217;, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function1,6-9.

The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD.

Valutazione del rene Zebrafish Funzione Con un test Clearance fluorescente

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal fluid accumulation and extracellular matrix formation. Activity of ion channels and intracellular calcium signaling are key physiologic parameters which determine functions of tubular epithelium. We developed a method suitable for real-time observation of ion channels activity with patch-clamp technique and registration of intracellular Ca2+ level in epithelial monolayers freshly isolated from renal cysts. PCK rats, a genetic model of autosomal recessive polycystic kidney disease (ARPKD), were used here for ex vivo analysis of ion channels and calcium flux. Described here is a detailed step-by-step procedure designed to isolate cystic monolayers and non-dilated tubules from PCK or normal Sprague Dawley (SD) rats, and monitor single channel activity and intracellular Ca2+ dynamics. This method does not require enzymatic processing and allows analysis in a native setting of freshly isolated epithelial monolayer. Moreover, this technique is very sensitive to intracellular calcium changes and generates high resolution images for precise measurements. Finally, isolated cystic epithelium can be further used for staining with antibodies or dyes, preparation of primary cultures and purification for various biochemical assays.

Ion channels expressed in renal tubular epithelium play a significant role in the pathology of polycystic kidney disease. Here we describe experimental protocols used to perform patch-clamp analysis and intracellular calcium level measurements in cystic epithelium freshly isolated from rodent kidneys.

Implementazione Patch Clamp e vivo microscopia a fluorescenza per il monitoraggio delle proprietà funzionali di recente isolato PKD Epithelium

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal ...

Use of 3D Robotic Ultrasound for In Vivo Analysis of Mouse Kidneys

Common modalities for in vivo imaging of rodents include positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Each method has limitations and advantages, including availability, ease of use, cost, size, and the use of ionizing radiation or magnetic fields. This protocol describes the use of 3D robotic US for in vivo imaging of rodent kidneys and heart, subsequent data analysis, and possible research applications. Practical applications of robotic US are the quantification of total kidney volume (TKV), as well as the measurement of cysts, tumors, and vasculature. Although the resolution is not as high as other modalities, robotic US allows for more practical high throughput data collection. Furthermore, using US M-mode imaging, cardiac function may be quantified. Since the kidneys receive 20%-25% of the cardiac output, assessing cardiac function is critical to the understanding of kidney physiology and pathophysiology.

Use of 3D Robotic Ultrasound for In Vivo Analysis of Mouse Kidneys

This protocol demonstrates robotic ultrasound (US) as a practical, cost-effective, and quick alternative to traditional non-invasive image modalities.

Uso di ultrasuoni robotici 3D per l'analisi in vivo dei reni di topo

Common modalities for in vivo imaging of rodents include positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), ...

Cytogenetics

Cytogenetics is the field of study devoted to chromosomes, and involves the direct observation of a cell&#8217;s chromosomal number and structure, together known as its karyotype. Many chromosomal abnormalities are associated with disease. Each chromosome in a karyotype can be stained with a variety of dyes to give unique banding patterns. More recent techniques, including comparative genomic hybridization and fluorescence in situ hybridization (FISH), allow for detecting specific chromosomal features or abnormalities.This video will begin by examining the principles of these classical and modern cytogenetics techniques. This is followed by an examination of a general protocol for performing FISH. Finally, several examples of how karyotyping can be applied to various medical applications are presented.

Cariotipo spettrale allo studio anomalie cromosomiche negli esseri umani e nei topi con malattia del rene policistico

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Rilevazione di Inter-aberrazioni cromosomiche stabili per fluorescenza multipla