Supportato da sovvenzioni da parte del Canadian Institutes of Health Research, il Canadian Stroke, e Alberta Heritage Foundation for Medical Research.

Rotorod camminare apparato Il rotorod era un cilindro rotante, 4 cm di diametro, fissato 35 centimetri dal suolo e chiuso da plexiglas trasparente. Il cilindro rotorod era coperta di rivestimento in gomma testurizzata, che facilita la trazione. Il pavimento dell&#39;apparato era coperto da uno strato di schiuma per evitare lesioni quando gli animali è caduto. Un piccolo motore elettrico fornito di alimentazione per accendere il rotorod tramite una cinghia di gomma. Il rotorod è stato impostato per ruotare una volta ogni 5 s. Registrazioni video e cinematica Ad alta velocità videocamera digitale è stata utilizzata per filmare gli animali, con un tempo di posa di un 1000 di secondo. Il frontale, laterale e posteriore viste sono state girate, mentre l&#39;animale stava camminando sul rotorod. A due bracci Nikon, fonte di luce fredda MII e un set di luci in fibra ottica sono stati utilizzati per fornire l&#39;illuminazione adeguata durante la registrazione. I nastri sono stati analizzati frame-by-frame utilizzando un Sony DSR 20 DV ponte CAM. Fotogrammi del video sono stati catturati con un frame grabber e computer Macintosh. Procedura di prova e analisi Al fine di valutare le differenze tra i movimenti a piedi del 6-OHDA e ratti di controllo, le registrazioni video da post-chirurgica giorni 15 - 30 (dopo l&#39;eventuale recupero dalla lesione ci si potrebbe aspettare di essere in gran parte completa) sono stati esaminati da due indipendenti osservatori ciechi alle condizioni sperimentali. Il nigrostriatale 6-OHDA lesione è stata indotta in dell&#39;emisfero destro, con conseguente deficit duratura nella sinistra anteriori e gli arti posteriori. Una scala di valutazione è stato sviluppato per tasso di postura dei topi &#39;ei movimenti passo degli arti anteriori (Whishaw et al., 2003). Cinque componenti di intensificare sono stati valutati: <ol><li> postura, la valutazione della postura è stata fatta esaminando la posizione del ratto sul tamburo; </li><li> ascensore e di rilascio, la parte del ciclo di un passo in cui è stata sollevata la zampa dal tamburo; </li><li> trasportare, la parte del ciclo di passo in cui si è tenuto l&#39;arto sopra il tamburo, come il topo spostato in avanti; </li><li> anticipo, la porzione di raggiungere, in cui è stato esteso l&#39;arto per ritrovare il contatto con il tamburo, e </li><li> collocamento, in cui è stata posta la zampa posteriore sul tamburo. </li></ol> I seguenti criteri sono stati utilizzati per definire un movimento normale: <ul><li> Postura: Il corpo del topo era orientata sul tamburo con la testa in su. </li><li> Sollevare e rilascio: Il, gomito spalla e polso flesso alzando la zampa dalla superficie del tamburo. </li><li> Carry: La zampa è tenuta al di sopra della superficie del tamburo, le cifre sono liberamente flesse, e le punte cifre sono centrati sulla linea mediana del corpo. Anticipo: L&#39;arto è esteso in avanti con le cifre in movimento da una posizione semiflessa ad una posizione esteso e aperto. </li><li> Posizionamento: L&#39;arto è esteso alla spalla, del gomito e del polso e le cifre sono aperti per posizionare la zampa in avanti e sulla superficie del tamburo, con 2 cifre (seconda cifra dalla linea mediana) che si trova circa al centro della di ratto il corpo. </li></ul> Ogni movimento è stato valutato su una scala a 3 punti: 1 = / il movimento simile a quello di un ratto di controllo, 0,5 = / il movimento è apparso un po &#39;anormale, e 0 = / il movimento era chiaramente anomala (Whishaw et al, 2003).. Questa scala di valutazione è stato poi utilizzato per valutare cinque movimenti un passo da ogni ratto nei giorni post-operatorio 1, 3, 5, 7, 9 e 15. I punteggi dei cinque misure sono state in media per ogni ratto ogni giorno, ed i risultati sottoposto ad un ANOVA con giornate come misura ripetuta. Panoramica delle rotorod camminare in 6-OHDA e ratti di controllo La scala di valutazione movimento è stato applicato al sessioni di test post-operatorio per l&#39;analisi del movimento qualitativo. Durante le sessioni di test, il 6-OHDA gruppo lesione ricevuto punteggi significativamente più bassi rispetto al gruppo di controllo, F (1,8) = 33,2, p &lt;0,001, ma non c&#39;era alcun effetto significativo della giornata di test, né vi era un gruppo significativo dall&#39;interazione giorni di test. I punteggi di intensificare sono stati poi analizzati separatamente con la zampa anteriore come una variabile (per la 6-OHDA ratti dell&#39;arto anteriore controlaterale rispetto alla lesione dell&#39;arto anteriore ipsilaterale alla lesione, per i ratti di controllo, la zampa sinistra contro la zampa destra). C&#39;è stato un effetto significativo del forelimb, F (1,40) = 15,8, p &lt;0.01, e ci fu una significativa interazione di gruppo da parte forelimb, F (1,40) = 15.0, p &lt;0,01. Follow-up t-test ha indicato che il controlaterale a lesione zampa anteriore del 6-OHDA gruppo ha ricevuto punteggi significativamente più bassi rispetto al ipsilaterale a zampa anteriore o lesione degli arti anteriori dei topi di controllo, che non differiva. Al fine di determinare quali componenti del movimento hanno contribuito al punteggio ottenuto dai poveri 6-OHDA gruppo, le misure della posturaed i singoli componenti del ciclo passo dalla arti anteriori ipsilaterale e controlaterale del 6-OHDA gruppo sono stati confrontati con i valori ottenuti dai topi di controllo. Il 6-OHDA gruppo differivano per postura e tutti i componenti passo da parte dell&#39;arto controlaterale, ma nessuno dei componenti dell&#39;arto omolaterale (p &lt;0,05). Differenze qualitative e descrittivi della postura sotto controllo e 6-OHDA ratti In un ratto di controllo, la testa è tenuto in posizione orizzontale, e la zampa anteriore di sostegno e arti posteriori si estendono al tamburo formando un arco di sostegno. I ratti 6-OHDA tipicamente camminava con la testa in giù con le loro arti anteriori compromessa (di fronte allo spettatore) posizionato più vicini e più in alto sulla superficie del tamburo che si è verificato per gli arti ipsilaterale o degli arti di ratti di controllo (vedi video di un topo di controllo con punteggio &quot;1&quot; per la postura, e un 6-OHDA animale lesione con un punteggio di &quot;0&quot;). Tipicamente, l&#39;anomalia della postura possono essere visto a colpo d&#39;occhio dalla posizione relativa della testa di un topo. Forelimb passo Nella sequenza di controllo, il passo è zampa anteriore sollevata, flettendo la, spalla gomito e polso. Come l&#39;arto viene sollevato, le cifre sono liberamente flesse, e l&#39;arto viene sollevato ben al di sopra della superficie del tamburo. Quando l&#39;arto è avanzata, si estende alle spalle, gomito e polso e come è collocato le cifre sono estese e aperte. Rispetto ai topi di controllo, l&#39;ampiezza di tutti i componenti del DA-impoverito ratti dell&#39;arto controlaterale alla lesione è attenuato. L&#39;arto rilascia il tamburo più tardi, viene sollevato di meno, ed è avanzato una distanza più breve è il controllo degli arti anteriori. Inoltre, quando l&#39;arto viene sollevato, le cifre sono flesse più, e quando l&#39;arto viene posto, le cifre sono estesi e aperti meno di quelli di un topo di controllo. La zampa anteriore di controllo viene sollevato una volta raggiunta la cima del tamburo, il polso è flesso per sollevare la zampa alto e in avanti, e il polso è esteso con l&#39;avanzare degli arti. Al contrario, la 6-OHDA ratto controlaterale alla lesione degli arti anteriori è portato avanti caudalmente dal movimento del tamburo, e rilascia così poi con il polso ancora esteso. Flessione del polso sembra essere passivo e si verifica come le cifre sono trasportati indietro dal movimento del tamburo, e quando dell&#39;arto anteriore è portato in avanti, la zampa e avambraccio non sono chiaramente sollevati dalla superficie del tamburo. La zampa anteriore di un topo di controllo si estende ben in avanti e le cifre aperto con l&#39;avanzare degli arti. Come l&#39;arto viene posto, è posizionato con un movimento arpeggio, con 5 cifre contatto con la superficie del primo tamburo, seguito successivamente da numeri 4, 3, e 2. Al contrario, l&#39;arto del 6-OHDA ratto è alzato ed esteso di meno, le cifre sono meno estesi, e contatto con la superficie del tamburo quasi in concomitanza con un &#39;schiaffo&#39;. Il video mostra passo-passo i movimenti degli arti anteriori in un animale di controllo che sono stati segnati come &quot;1&quot;, i movimenti degli arti anteriori e passo in un 6-OHDA animale lesione che sono stati principalmente punteggio di &quot;0&quot;. Degli arti posteriori passo C&#39;erano anche menomazioni agli arti posteriori controlaterale a lesione del 6-OHDA ratti. Come l&#39;arto di controllo posteriori si muove all&#39;indietro, sembra spingere contro il tamburo che ruota e come lo fa, le dita ruotare verso l&#39;esterno. Come l&#39;arto si muove in avanti, il piede è oscillato verso l&#39;esterno. L&#39;arto posteriore del 6-OHDA ratto si muove direttamente indietro, con rotazione del tamburo e se non altro, svolta leggermente verso l&#39;interno. Come l&#39;arto si muove in avanti, il piede è portato direttamente in avanti. Il piede di altalene ratto di controllo verso l&#39;esterno e come è avanzata, le dita si tengono superiore alla caviglia e le dita dei piedi estendere. Durante il posizionamento del ratto controllo, punta 5 contatti sulla superficie del tamburo primo e il peso è distribuito attraverso le dita dei piedi e il palmo. Al contrario, le dita del 6-OHDA ratto sono flesse durante trasportare e il posizionamento è fatto senza un movimento arpeggio. Le dita del ratto di controllo vengono rilasciati in un modello invertito arpeggio come toe 5 è sollevata prima e punta 1 è sollevato scorso. Questo movimento mancava nel 6-OHDA ratto lesione alla caviglia, mentre ha mantenuto la sua postura flessa durante il movimento di trasporto. Ancora una volta i movimenti prima del 6-OHDA ratto sono attenuate, con l&#39;arto inferiore exorotated, sollevato di meno, e avanzato inferiore a quella del ratto di controllo. Come ratti di controllo posto le dita dei piedi, sono estesi e aperti, in modo tale che punta 5 punti lateralmente rispetto al corpo del ratto. Toe 5 contatti del tamburo primo seguito da le dita restanti in modo che il topo sostenuto il suo peso sulle punte dei piedi. Per la 6-OHDA ratto, le dita sono un po &#39;flesse come il piede è portato avanti, le dita sono più bassi rispetto a orizzontale fino alla caviglia, c&#39;è meno apertura delle dita quando il piede è posto sul tamburo. Lunghezza del passo è anche un po &#39;più breve di quella dei topi di controllo. Al fine di quantificare le differenze tra i control e 6-OHDA movimenti degli arti posteriori, le componenti di ascensore e di rilascio, trasporto, promozione, posizionamento e stati valutati su una scala a 3 punti, così come le arti anteriori. Le misure sono state fatte da registrazioni video effettuate giorno dopo chirurgici 20, e tre cicli di passo sono stati valutati per ogni ratto. Ci sono state differenze significative del gruppo, F (1,8) = 47.6, p &lt;0,001. Follow-up t-test sui componenti indicato che tutti i componenti dei topi di controllo ha ricevuto punteggi significativamente più alti di quanto facessero le componenti della 6-OHDA ratti. Il video mostra i movimenti degli arti posteriori passo in un animale di controllo che sono stati segnati come &quot;1&quot;. Dell&#39;arto posteriore passo i movimenti nel 6-OHDA mostrato nel video sono state principalmente valutate con un punteggio di &quot;0&quot;.

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Lo scopo del presente studio era di esaminare se il test di abilità rotorod roditori locomozione può essere utilizzato per l&#39;esame qualitativo della postura, passo zampa anteriore e degli arti posteriori intervenendo ratti sistema nervoso feriti. Per l&#39;analisi, i ratti con unilaterale svuotamenti DA e ratti di controllo sono stati video registrati dalla vista frontale, laterale e posteriore. Una scala di valutazione della postura e dei movimenti degli arti anteriori hanno indicato che i movimenti passo erano cronicamente compromes...

A correction was made to: <a href="http://www.jove.com/video/1030/">Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat</a>. An additional paragraph was added to the discussion, the abstract was modified, and another reference was included.
Additional reference:
15. Whishaw IQ, Li K, Whishaw PA, Gorny B, Metz GA. Distinct forelimb and hind limb stepping impairments in unilateral dopamine-depleted rats: use of the rotorod as a method for the qualitative analysis of skilled walking. J Neurosci Meth 126, 13-23 (2003).
Revised Abstract:
High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.
Additional Discussion Paragraph:
A more detailed presentation of the present study has been made (Whishaw et al, 2003), but the present study presents the video support describing the stepping movement in the good and affected limbs of unilateral dopamine-depleted rats. For the analysis, rats with unilateral DA depletions and control rats were video recorded from front, lateral and posterior views. A rating scale of posture and forelimb movements indicated that stepping movements were chronically impaired following surgery. Examination of limb movements indicated that whereas the DA-depleted rats could use the limbs contralateral to the lesion for support, they received minimal use for shifting weight. The results of this study indicate that the rotorod task, in addition to providing quantitative measures of motor impairments, can also provide insights into the qualitative impairments.
Original Abstract:
The rotorod test, in which animals walk on a rotating drum, is widely used to assess motor status in laboratory rodents. Performance is measured by the duration that an animal stays up on the drum as a function of drum speed. Here we report that the task provides a rich source of information about qualitative aspects of walking movements. Because movements are performed in a fixed location, they can readily be examined using high-speed video recording methods. The present study was undertaken to examine the potential of the rotorod to reveal qualitative changes in the walking movements of hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). Beginning on the day following surgery and then periodically over the next two months, the rats were filmed from frontal, lateral, and posterior views as they walked on the rotorod. Behavior was analyzed by frame-by-frame replay of the video records. Rating scales of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in their posture and in the use of the limbs contralateral to the DA-depletion. The contralateral limbs not only displayed postural and movement abnormalities, they participated less in initiating and sustaining propulsion than did the ipsilateral limbs. These findings not only reveal new deficits secondary to unilateral DA-depletion, but also show that the rotorod can provide a robust tool for the qualitative analysis of movement.

A correction was made to: <a href="http://www.jove.com/video/1030/">Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat</a>. An additional paragraph was added to the discussion, the abstract was modified and another reference was included.

Erratum: Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

use of rotorod as a method for the qualitative analysis of walking in rat

Il test rotorod, in cui gli animali camminare su un tamburo rotante, è ampiamente usato per valutare lo stato del motore nei roditori da laboratorio. La performance è misurata dalla durata che un animale rimane sul tamburo in funzione della velocità del tamburo. Qui si segnala che il compito fornisce una ricca fonte di informazioni su aspetti qualitativi dei movimenti a piedi. Perché i movimenti sono eseguiti in una posizione fissa, possono facilmente essere esaminati utilizzando ad alta velocità, metodi di registrazione video. Il presente studio è stato intrapreso per esaminare il potenziale del rotorod per rivelare cambiamenti qualitativi nei movimenti a piedi di emi-Parkinson ratti analogico, ottenuta per iniezione di 6-idrossidopamina (6-OHDA) nel fascio diritto nigrostriatale ad esaurire dopamina nigrostriatale (DA ). A partire dal giorno successivo l&#39;intervento chirurgico e poi periodicamente nel corso dei prossimi due mesi, i ratti sono stati girati da vista frontale, laterale e posteriore mentre camminavano sul rotorod. Comportamento è stato analizzato fotogramma per fotogramma per fotogramma riproduzione delle registrazioni video. Scale di valutazione di intensificare comportamento indicato che l&#39;emi-Parkinson ratti erano cronicamente alterata nella loro postura e l&#39;uso degli arti controlaterali al DA-esaurimento. Gli arti controlaterale non solo visualizzati anomalie posturali e di movimento, hanno partecipato meno di avviare e sostenere la propulsione che ha gli arti omolaterali. Questi risultati non solo rivela nuovi disavanzi unilaterale secondaria a DA-esaurimento, ma mostrano anche che il rotorod in grado di fornire uno strumento valido per l&#39;analisi qualitativa del movimento.

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Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

Il test rotorod viene usato per valutare lo stato del motore nel movimento dei piedi emi-Parkinson ratti analogico.

L&#39;uso di Rotorod come un metodo per l&#39;analisi qualitativa del Camminare in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

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12.9K Views.  University of Lethbridge. Il test rotorod viene usato per valutare lo stato del motore nel movimento dei piedi emi-Parkinson ratti analogico.

Video: Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pirosequenziamento: un metodo semplice per genotipizzazione accurata

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

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Biologia

The Structure of Skilled Forelimb Reaching in the Rat: A Movement Rating Scale

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and primates. The video describes a test situation that allows filming of repeated acts of reaching for food by the rat that has been mildly food deprived. A rat is trained to reach through a slot in a holding box for food pellet that it grasps and then places in its mouth for eating. Reaching is accomplished in the main by proximally driven movements of the limb but distal limb movements are used for pronating the paw, grasping the food, and releasing the food into the mouth. Each reach is divided into at least 10 movements of the forelimb and the reaching act is facilitated by postural adjustments. Each of the movements is described and examples of the movements are given from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation. Because the reaching act for the rat is very similar to that displayed by humans and nonhuman primates, the scale can be used for comparative purposes. from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation.

Experiments on animals were performed in accordance with the guidelines and regulations set forth by the University of Lethbridge Animal Care Committee in accordance with the regulations of the Canadian Council on Animal Care.

The skilled reaching scale divides the movement by a forelimb in a reach for food act into composite elements each of which are evaluated with a three-point scale. The rating scale is described for a normal rat and can be applied toward evaluating neurological motor disorders.

La struttura della zampa anteriore Skilled Raggiungere nel Ratto: una scala di valutazione Movimento

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and ...

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

Il ramo Ladder Walking attività: un sistema di punteggio e la sua applicazione pratica.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

Analisi dello sviluppo di un fenotipo morfologico come funzione della concentrazione di proteine ​​in lievito

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

Microiniezione di embrioni Medaka per essere utilizzato come organismo modello genetico

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

Un metodo semplice per l&#39;imaging Arabidopsis Foglie Utilizzando Perfluorodecalin come Media Imaging infiltrativa

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

L&#39;uso di chemostati in Biologia microbica Sistemi

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

Un ibrido metodo di estrazione del DNA per la valutazione delle comunità batteriche qualitativa e quantitativa di pollame campioni di produzione

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

Prelievo di sangue dalla vena della coda laterale del Topo

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...