Questa ricerca è stata sostenuta dalle concessioni dal Medical Research Council, le scienze naturali e ingegneria Research Council del Canada, e il Canadian Stroke. GAM è stata sostenuta anche dalla Alberta Heritage Foundation for Medical Research.

Scala a pioli camminando apparecchiatura di prova Il ramo scala orizzontale a piedi apparecchiatura di prova consisteva di pareti laterali in plexiglas trasparente e gradini in metallo (3 mm di diametro), che potrebbe essere inserito per creare un pavimento con una distanza minima di 1 cm tra i pioli (vedi Fig. 1;. Metz e Whishaw , 2003). Le pareti laterali sono stati 1 m di lunghezza e 19 cm di altezza misurata dalla altezza della pioli. La scala è stata elevata 30 cm sopra il terreno con una gabbia di partenza neutro e un rifugio (gabbia di casa) alla fine. Perché gli animali sono stati abituati durante l&#39;allenamento, l&#39;elevazione degli apparati è stata poche probabilità di causare ansia. La larghezza del vicolo è stato adattato alle dimensioni dell&#39;animale, in modo che fosse di circa 1 cm più largo di un animale per evitare che l&#39;animale da voltarsi. La difficoltà del compito è stata modificata variando la posizione dei pioli metallici. Un andamento regolare dei pioli permesso agli animali di imparare il modello più sessioni di formazione diversi e di anticipare la posizione dei pioli (Fig. 1, modello A). Un modello irregolare che è stato cambiato da prova a prova impedito all&#39;animale di imparare il disegno (Fig. 1, modello B). Per la disposizione regolare, i pioli sono distanziati ad intervalli di 2 cm. Per l&#39;irregolare, la distanza dei pioli varia sistematicamente da 1 a 5 cm. Cinque modelli di modelli gradino irregolari sono stati utilizzati, in modo che gli stessi modelli sono stati applicati a tutti gli animali di standardizzare la difficoltà della prova e migliorare la comparabilità dei risultati (vedi risultati). Videoregistrazione Una macchina fotografica (Canovision, Canon Inc.) è stata posizionata con una leggera angolazione ventrale, in modo da posizioni di tutti e quattro gli arti possono essere registrate simultaneamente. La velocità dell&#39;otturatore è stato fissato a 500 - 2000 s. Le videoregistrazioni sono stati analizzati usando frame-by-frame di analisi a 30 f / sec. Formazione comportamentale e analisi di test Gli animali sono stati addestrati ad attraversare la scala da una gabbia neutrale per raggiungere il loro gabbia casa, quindi la gabbia a casa con fratellini fornito il rinforzo positivo per camminare. Tutti gli animali attraversato la scala nella stessa direzione. Nessun ulteriore rafforzamento è stata data per motivare gli animali ad attraversare la scala. Tutti gli animali sono stati addestrati e testati cinque volte per sessione. Colpa punteggio piede La valutazione qualitativa del posizionamento degli arti posteriori e la zampa anteriore è stata effettuata utilizzando un piede sistema di punteggio di guasto come descritto in precedenza (Metz e Whishaw, 2003). L&#39;analisi è stata fatta con l&#39;ispezione delle registrazioni video fotogramma per fotogramma. A pochi passi consecutivi di ogni arto sono stati analizzati. Pertanto, l&#39;ultimo passo prima di una interruzione del cammino, come ad esempio un arresto o un guasto del piede, e il primo passo dopo un&#39;interruzione non sono stati segnati. Il ciclo di stepping ultimo eseguito alla fine della scala è stata esclusa dal punteggio. Posizionamento degli arti è stato segnato, in termini di posizionamento degli arti su un gradino e protrusione degli arti tra pioli quando una miss si è verificato. I tipi di posizionamento piedi o zampa sui gradini sono stati valutati utilizzando un 7-categoria di scala (vedi fig. 2). La posizione dei piedi o zampa sul gradino è stata valutata in base alla loro posizione e gli errori che si sono verificati nel posizionamento di precisione. (0) Totale perdere. 0 punti sono stati dati quando l&#39;arto completamente saltato un gradino, cioè non lo tocco, e una caduta si è verificato. Una caduta è stata definita come un arto profondamente caduta in-tra pioli e la postura e l&#39;equilibrio sono stati disturbati. (1) scivolamento profondo. L&#39;arto è stato inizialmente posto su un gradino, poi sfilò quando il peso-cuscinetto e ha causato una caduta. (2) scivolare leggermente. L&#39;arto è stato posto su un ramo, quando sfilò sostenere il peso, ma non provocare una caduta, né interrompere il ciclo del passo. In questo caso, l&#39;animale era in grado di mantenere l&#39;equilibrio e continuare a un&#39;andatura coordinato. (3) sostituzione. L&#39;arto è stato posto su un ramo, ma prima che fosse il carico è stato rapidamente sollevato e posto su un altro gradino. (4) Correzione. L&#39;arto volti per un gradino, ma è stato poi messo su un altro gradino, senza toccare il primo. In alternativa, un punteggio di 4 è stato registrato, se un arto è stato posto su un ramo ed è stato subito riposizionato, pur rimanendo sul gradino stesso. (5) collocamento parziale. L&#39;arto è stato posto su un ramo con entrambi i polsi o le cifre della zampa anteriore e tallone o le dita delle arti posteriori. (6) Il corretto posizionamento. La porzione mediana del palmo di un arto è stato immesso sul gradino con il supporto peso. Quando diversi errori avvenuti nello stesso momento, il più basso dei punteggi è stato registrato. Per esempio, se un piede è stato posto su un gradino e poi messo su un altro nello stesso passaggio (punteggio 3), e poi scivolato ed è caduto nel mezzo pioli (punteggio 1), un punteggio di 1 è stato registrato. When una caduta avvenuta, solo l&#39;arto di iniziare l&#39;errore è stato valutato e nessuna delle altre membra è stato segnato fino a quando l&#39;animale aveva riposizionato tutti gli arti. Punteggi errore di cinque studi sono stati in media per l&#39;analisi. Piede accuratezza analisi collocazione (numero di errori) Il numero di errori in ogni incrocio è stato contato. Gli errori sono stati determinati sulla base del sistema di punteggio piedi colpa. Un errore è stato definito come ogni posizionamento degli arti che hanno ricevuto un punteggio di punti 0, 1 o 2, cioè un errore rappresenta alcun tipo di scivolare a piedi o perdere totale. Il numero di errori e il numero di passaggi è stato registrato per ogni arto separatamente. Da questi dati, il numero medio di errori per ogni passo è stato calcolato e una media di cinque prove. Il secondo parametro quantitativo è stato analizzato il tempo medio necessario per attraversare tutta la lunghezza del compito scala. Misurazione del tempo ha iniziato dopo che l&#39;animale è stato posto sulla scala e cominciò a camminare per l&#39;intera lunghezza di essa. Il tempo trascorso in un animale una sosta non è stata inclusa nella misurazione. Zampe anteriori punteggio cifre Il punteggio zampe anteriori cifra è stata registrata quando la zampa è stato posto correttamente con la sua porzione mediana su un ramo (ovvero punteggio 6 nel sistema di punteggio piede errore). La misura in cui le cifre della zampa anteriore può essere piegato intorno ad un gradino è stato valutato in una scala di tre punti (Fig. 2). Il punteggio è stato applicato alla posizione con il supporto peso massimo, cioè la zampa anteriore è stato posizionato in verticale su un ramo. I punteggi sono stati dati come segue: (0) cifre chiuso in un approssimativo di 90 gradi. (1) chiuso in cifre di circa 45 gradi. (2) cifre completamente flesso in tutto il ramo. Decine di cinque passi sono stati mediati e utilizzati per l&#39;analisi. Figura leggende <img src="/files/ftp_upload/old/1204_2009_1_20_6/Fig 1 Grid.png" alt="figura 1" /> Fig. 1. Il ramo scala qualificati camminare apparecchiatura di prova in vista frontolateral, con le misure specifiche dell&#39;apparato indicato. Due modelli gradino differenti sono stati utilizzati nel presente studio: Modello A, arrangiamento gradino regolare, modello B, disposizione gradino irregolare. La disposizione gradino irregolari variano tra prove consecutive. <img src="/files/ftp_upload/old/1204_2009_1_20_7/Fig 2 (angle lines).png" alt="figura 2" /> Fig. 2. Rappresentante fotografie che illustrano le tre categorie del punteggio cifra. Si noti l&#39;angolo delle cifre in relazione alle arti anteriori quando nel sostenere il peso totale.

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Il presente studio presenta il gradino scala a piedi un&#39;attività come un nuovo test per valutare a piedi qualificati, il posizionamento degli arti e nella coordinazione degli arti. Il ramo scala camminare attività consente discriminazioni tra i sottili disturbi della funzione motoria, combinando analisi qualitativa e quantitativa di camminare qualificati. La procedura di analisi video può essere utilizzato per un resoconto più descrittivo dei danni, e la scala di valutazione semplice, descritto in questo documen...

the ladder rung walking task: a scoring system and its practical application.

Progressi nello sviluppo di modelli animali per / ictus, lesioni del midollo spinale, e di altre malattie neurodegenerative richiede test ad alta sensibilità per elaborare aspetti distinti della funzione motoria e per determinare la perdita anche di sottile capacità di movimento. Per migliorare l&#39;efficacia e la risoluzione dei test, i test dovrebbero consentire misure qualitative e quantitative della funzione motoria e di essere sensibile ai cambiamenti nelle prestazioni durante i periodi di recupero. Il presente studio descrive un nuovo compito per valutare camminare abile nel ratto di misurare sia la zampa anteriore e la funzione degli arti posteriori, allo stesso tempo. Gli animali sono tenuti a camminare lungo una scala orizzontale su cui la distanza tra i gradini è variabile e viene periodicamente cambiata. Variazioni nella spaziatura gradino evitare che gli animali imparando la posizione assoluta e relativa dei pioli e quindi minimizzare la capacità degli animali di compensare la disabilità attraverso l&#39;apprendimento. Inoltre, cambiando la distanza tra i pioli consente la prova di essere utilizzato più volte in studi a lungo termine. I metodi sono descritti per la descrizione sia quantitativi che qualitativi di prestazioni anteriori e degli arti posteriori, tra cui arti collocare, passo-passo, la coordinazione. Inoltre, l&#39;uso di strategie di compensazione è indicata da passi falsi o fasi di compensazione in risposta a un altro smarrimento dell&#39;arto.

Watch this Scientific Journal Video about The Ladder Rung Walking Task: A Scoring System and its Practical Application. at JoVE.com

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

Il ramo scala camminare compito è un nuovo test per valutare a piedi qualificati e misurare sia degli arti posteriori e la zampa anteriore collocamento, passo-passo, e tra arti coordinamento.

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 ...

the-ladder-rung-walking-task-scoring-system-its-practical

Research

JoVE Journal

Biology

25.8K Views.  University of Lethbridge. Il ramo scala camminare compito è un nuovo test per valutare a piedi qualificati e misurare sia degli arti posteriori e la zampa anteriore collocamento, passo-passo, e tra arti coordinamento.

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

Use of Rotorod as a Method for the Qualitative Analysis of Walking 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 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.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

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 ...

Ricerca

Biologia

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

Lens trapianto in Zebrafish e la sua applicazione nell&#39;analisi dei Mutanti Eye

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

Visualizzazione di Caenorhabditis Elegans Strutture cuticola Utilizzando il Dye lipofili Vital, Dii

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. It is likely to play a role in preventing accumulation of recombinogenic intermediates that would otherwise accumulate at transiently stalled replication forks, consistent with a hyper-recombinant phenotype of cells lacking WRN. In humans, the exonuclease domain comprises an N-terminal portion of a much larger protein that also possesses helicase activity, together with additional sites important for DNA and protein interaction. By contrast, in Drosophila, the exonuclease activity of WRN (DmWRNexo) is encoded by a distinct genetic locus from the presumptive helicase, allowing biochemical (and genetic) dissection of the role of the exonuclease activity in genome stability mechanisms. Here, we demonstrate a fluorescent method to determine WRN exonuclease activity using purified recombinant DmWRNexo and end-labeled fluorescent oligonucleotides. This system allows greater reproducibility than radioactive assays as the substrate oligonucleotides remain stable for months, and provides a safer and relatively rapid method for detailed analysis of nuclease activity, permitting determination of nuclease polarity, processivity, and substrate preferences.

Exonucleases play critical roles in ensuring genome stability. Loss of WRN exonuclease function results in premature aging. Studying substrates and other requirements of the nuclease in vitro can help elucidate its role in vivo. Here we demonstrate a rapid and reproducible fluorescence-based assay to measure its nuclease activity.

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. ...

A Practical Guide to Phylogenetics for Nonexperts

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this topic and so it presents inherent problems. Here we compile a practical introduction to phylogenetics for nonexperts.&#160;We outline in a step-by-step manner, a pipeline for generating reliable phylogenies from gene sequence datasets. We begin with a user-guide for similarity search tools via online interfaces as well as local executables. Next, we explore programs for generating multiple sequence alignments followed by protocols for using software to determine best-fit models of evolution. We then outline protocols for reconstructing phylogenetic relationships via maximum likelihood and Bayesian criteria&#160;and finally describe tools for visualizing phylogenetic trees. While this is not by any means an exhaustive description of phylogenetic approaches, it does provide the reader with practical starting information on key software applications commonly utilized by phylogeneticists. The vision for this article would be that it could serve as a practical training tool for researchers embarking on phylogenetic studies&#160;and also serve as an educational resource that could be incorporated into a classroom or teaching-lab.

Here we describe a step-by-step pipeline for generating reliable phylogenies from nucleotide or amino acid sequence datasets. This guide aims to serve researchers or students new to phylogenetic analysis.&#160;

Una guida pratica per Phylogenetics per i non esperti

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

Progettazione e analisi del comportamento delle preferenze di temperatura e del suo ritmo circadiano in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&#38;E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution.
This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

Ricostruzione di 3-Dimensional Volume Istologia e la sua applicazione per studiare mouse ghiandole mammarie

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Imaging Riorganizzazione spaziale di una via di segnalazione MAPK Utilizzando il sistema transitorio Expression Tabacco

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive organs/gametes. Recent advances in CRISPR-Cas9 genome editing technology allow for high editing efficacy and timesaving knockout mutations of endogenous candidate genes at specific sites. Additionally, Agrobacterium-mediated genetic transformation of rice is also a key method for gene modification, which has been widely adopted by many public and private laboratories. In this study, we applied CRISPR-Cas9 genome editing tools and successfully generated three male sterile mutant lines by targeted genome editing of OsABCG15 in a japonica cultivar. We used a modified Agrobacterium-mediated rice transformation method that could provide excellent means of genetic emasculation for hybrid seed production in rice. Transgenic plants can be obtained within 2&#8211;3 months and homozygous transformants were screened by genotyping using PCR amplification and Sanger sequencing. Basic phenotypic characterization of the male sterile homozygous line was performed by microscopic observation of the rice male reproductive organs, pollen viability analysis by iodine potassium iodide (I2-KI) staining semi-thin cross-sectioning of developing anthers.

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

This work describes the use of CRISPR-Cas9 genome editing technology to knockout endogenous gene OsABCG15 followed by a modified Agrobacterium-mediated transformation protocol to produce a stable male-sterile line in rice.

Agrobacterium-Mediato Trasformazione Genetica, Produzione Transgenica, e la sua applicazione per lo studio dello sviluppo riproduttivo maschile nel riso

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive ...

A Simple Dry Sectioning Method for Obtaining Whole-Seed-Sized Resin Section and Its Applications

The morphology, size and quantity of cells, starch granules&#160;and protein bodies in seed determine the weight and quality of seed. They are significantly different among different regions of seed. In order to view the morphologies of cells, starch granules and protein bodies clearly, and quantitatively analyze their morphology parameters accurately, the whole-seed-sized section is needed. Though the whole-seed-sized paraffin section can investigate the accumulation of storage materials in seeds, it is very difficult to quantitatively analyze the morphology parameters of cells and storage materials due to the low resolution of the thick section. The thin resin section has high resolution, but the routine resin sectioning method is not suitable to prepare the whole-seed-sized section of mature seeds with a large volume and high starch content. In this study, we present a simple dry sectioning method for preparing the whole-seed-sized resin section. The technique can prepare the cross and longitudinal whole-seed-sized sections of developing, mature, germinated, and cooked seeds embedded in LR White resin, even for large seeds with high starch content. The whole-seed-sized section can be stained with fluorescent brightener 28, iodine, and Coomassie brilliant blue R250 to specifically exhibit the morphology of cells, starch granules, and protein bodies clearly, respectively. The image obtained can also be analyzed quantitatively to show the morphology parameters of cells, starch granules, and protein bodies in different regions of seed.

This technique allows for the fast and simple preparation of whole-seed-sized resin section for the observation and analysis of cells, starch granules, and protein bodies in different regions of the seed.