Cette recherche a été financée par des subventions du Conseil de recherches médicales, en sciences naturelles et en génie du Canada, et le Réseau canadien contre les accidents. GAM a également été soutenu par l&#39;Alberta Heritage Foundation for Medical Research.

Réseau de contacts appareil de test de marche Le barreau d&#39;échelle horizontale marcher appareil d&#39;essai se composait de parois latérales en plexiglas clair et barreaux métalliques (3 mm de diamètre), qui pourrait être inséré pour créer un plancher avec une distance minimale de 1 cm entre les barreaux (voir la figure 1;. Metz et Whishaw , 2003). Les parois latérales étaient de 1 m de long et 19 cm de haut mesuré à partir de la hauteur des barreaux. L&#39;échelle a été surélevée de 30 cm au-dessus du sol avec une cage démarrage neutre et un refuge (cage) à la fin. Parce que les animaux ont été habitués pendant la formation, l&#39;élévation de l&#39;appareil était susceptible de provoquer l&#39;angoisse. La largeur de l&#39;allée a été ajusté à la taille de l&#39;animal, de sorte qu&#39;il a été d&#39;environ 1 cm plus large que d&#39;un animal pour empêcher l&#39;animal de se retourner. La difficulté de la tâche a été modifiée en faisant varier la position des barreaux métalliques. Un schéma régulier des barreaux a permis aux animaux pour apprendre la séquence au cours de plusieurs sessions de formation et d&#39;anticiper la position des barreaux (Fig. 1, Schéma A). Un motif irrégulier qui a été changé d&#39;un essai à empêcher l&#39;animal de l&#39;apprentissage du modèle (Fig. 1, Motif B). Pour l&#39;arrangement régulier, les barreaux sont espacés à intervalles de 2 cm. Pour le motif irrégulier, la distance des échelons variés systématiquement à partir de 1 à 5 cm. Cinq modèles de motifs irréguliers sonné ont été utilisés, de sorte que les mêmes modèles ont été appliqués à tous les animaux de normaliser la difficulté de l&#39;épreuve et d&#39;améliorer la comparabilité des résultats (voir résultats). Enregistrement vidéo Une caméra (Canovision, Canon Inc) a été positionné à un angle léger ventrale, alors les positions des quatre membres pourraient être enregistrées simultanément. La vitesse d&#39;obturation a été fixé à 500 - 2000 s. Les enregistrements vidéo ont été analysées en utilisant image-par-cadre d&#39;analyse à 30 f / sec. Formation comportementale et une analyse de test Les animaux ont été formés pour traverser l&#39;échelle d&#39;une cage neutre pour atteindre leur cage d&#39;origine, donc la cage à la maison avec la même portée à condition que le renforcement positif pour la marche. Tous les animaux croisés sur l&#39;échelle dans la même direction. Aucun renfort supplémentaire a été donnée pour motiver les animaux de traverser l&#39;échelle. Tous les animaux ont été formés et testés cinq fois par session. Notation faute de pied L&#39;évaluation qualitative de placement des membres antérieurs et postérieurs a été réalisée en utilisant un système de notation faute de pied comme décrit précédemment (Metz et Whishaw, 2003). L&#39;analyse a été faite par l&#39;inspection des enregistrements vidéo image par image. Seuls les étapes consécutives de chaque membre ont été analysés. Par conséquent, la dernière étape avant une interruption de la marche, comme un arrêt ou une faute de pied, et la première étape après une interruption ne sont pas marqués. Le cycle de l&#39;intensification dernière effectuée à la fin de l&#39;échelle a également été exclus de la notation. Le placement Limb a été marqué en termes de placement des membres sur une protubérance sonné et un membre entre les barreaux quand un manque s&#39;est produit. Les types de placement des pieds ou de pattes sur les barreaux ont été évalués en utilisant une échelle de 7 catégories (voir Fig. 2). Le placement des pieds ou de pattes sur le barreau a été classé en fonction de leur position et les erreurs survenues dans la précision de placement. (0) Total des manquer. 0 points ont été donnés lorsque le membre complètement raté un échelon, c&#39;est à dire ne pas le toucher, et une chute s&#39;est produite. Une chute a été définie comme une branche profondément tomber entre barreaux et la posture et l&#39;équilibre ont été perturbés. (1) de glissement profond. Le membre a d&#39;abord été placé sur un barreau, puis a glissé lors de port de poids et a causé une chute. (2) glisse légèrement. Le membre a été placé sur un échelon, a glissé de port de poids quand, mais n&#39;ont pas abouti à une chute, ni interrompre le cycle de la marche. Dans ce cas, l&#39;animal a été en mesure de maintenir l&#39;équilibre et de continuer une démarche coordonnée. (3) de remplacement. Le membre a été placé sur un échelon, mais avant qu&#39;elle portait le poids qu&#39;il a été rapidement levé et placé sur un autre échelon. (4) Correction. Le membre visant à un barreau, mais a ensuite été placé sur un autre barreau, sans toucher le premier. Alternativement, un score de 4 a été enregistré, si un membre a été placée sur un barreau et a été rapidement repositionné tout en restant sur le même barreau. (5) le placement partiel. Le membre a été placée sur un barreau soit avec le poignet ou les chiffres du membre antérieur ou du talon ou des orteils du membre postérieur. (6) mise en place correcte. La partie médiane de la paume d&#39;un membre a été placé sur l&#39;échelon avec le soutien de poids. Lorsque des erreurs différents survenus au même moment, le plus bas des scores a été enregistré. Par exemple, si un pied a d&#39;abord été placée sur un barreau, puis placé sur un autre dans la même étape (score 3), puis a glissé et est tombé entre-deux échelons (score 1), un score de 1 a été enregistré. Whfr d&#39;une chute survenue, seuls les membres initier l&#39;erreur a été évalué et aucun des autres membres a été marqué jusqu&#39;à ce que l&#39;animal avait repositionné tous les membres. Scores d&#39;erreur de cinq essais ont été en moyenne pour l&#39;analyse. Pied analyse de la précision de placement (nombre d&#39;erreurs) Le nombre d&#39;erreurs dans chaque croisement a été compté. Des erreurs ont été déterminés sur la base des système de notation faute de pied. Une erreur a été défini comme chaque placement membre qui a reçu un score de points 0, 1 ou 2, c&#39;est à dire une erreur représente aucune sorte de glissement du pied ou de manquer total. Le nombre d&#39;erreurs et le nombre d&#39;étapes a été enregistré pour chaque membre séparément. A partir de ces données, le nombre moyen d&#39;erreurs par étape a été calculé et la moyenne des cinq essais. Le second paramètre a été analysé quantitative le temps moyen nécessaire pour traverser toute la longueur de la tâche échelle. Mesure du temps a commencé après que l&#39;animal a été placé sur l&#39;échelle et a commencé à marcher toute la longueur de celui-ci. Le temps a passé un animal dans un arrêt n&#39;a pas été inclus dans la mesure. Patte score de chiffres Le score patte chiffres a été enregistrée lorsque la patte a été correctement placé sur la partie médiane de son d&#39;un cran (score soit 6 dans le système de notation faute de pied). La mesure dans laquelle les chiffres de la patte avant pourrait être fléchie autour d&#39;un barreau a été classé dans une échelle de trois points (Fig. 2). Le score a été appliquée à la position avec le soutien de poids au maximum, c&#39;est à dire la patte avant a été positionné à la verticale sur un barreau. Les scores ont été donnés comme suit: (0) Chiffres fermés dans une approximative de 90 degrés d&#39;angle. (1) Chiffres fermés dans un angle d&#39;environ 45 degrés. (2) chiffres complètement fléchie autour du barreau. Les scores des cinq étapes ont été moyennées et utilisées pour l&#39;analyse. Légendes des figures <img src="/files/ftp_upload/old/1204_2009_1_20_6/Fig 1 Grid.png" alt="Figure 1" /> Figure 1. Le barreau d&#39;échelle qualifiée de marche appareil d&#39;essai dans la vue frontolateral, avec les mesures spécifiques de l&#39;appareil indiqué. Deux modèles différents ont été utilisés sonné dans la présente étude: Un modèle, l&#39;arrangement sonne régulièrement, B Motif, l&#39;arrangement sonné irrégulière. L&#39;arrangement sonné irrégulière ont varié entre les essais consécutifs. <img src="/files/ftp_upload/old/1204_2009_1_20_7/Fig 2 (angle lines).png" alt="Figure 2" /> Fig. 2. Photographies illustrant représentant les trois catégories du score de chiffres. Notez l&#39;angle des chiffres par rapport à la patte avant quand à l&#39;appui de poids totale.

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La présente étude introduit les barreaux d&#39;une échelle de marche tâche comme un nouveau test pour évaluer la marche qualifiés, le placement des membres et des membres de la coordination. Le barreau d&#39;échelle marcher tâche permet la discrimination entre les perturbations subtiles de la fonction motrice, en combinant l&#39;analyse qualitative et quantitative de la marche qualifiés. La procédure d&#39;analyse vidéo peut être utilisée pour un compte rendu plus descriptive des déficiences, et l&#39;éch...

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

Progrès dans le développement de modèles animaux pour / accident vasculaire cérébral, traumatisme médullaire, et d&#39;autres maladies neurodégénératives nécessite des tests de haute sensibilité pour développer des aspects distincts de la fonction motrice et de déterminer la perte de la capacité, même subtile mouvement. Afin de renforcer l&#39;efficacité et la résolution des tests, des tests devraient permettre des mesures qualitatives et quantitatives de la fonction motrice et être sensible aux changements dans la performance pendant les périodes de récupération. La présente étude décrit une nouvelle tâche d&#39;évaluer la marche qualifiés chez le rat afin de mesurer à la fois des membres antérieurs et postérieurs de fonction au même moment. Les animaux sont nécessaires pour marcher le long d&#39;une échelle horizontale sur laquelle l&#39;espacement des barreaux est variable et est changé périodiquement. Les changements dans écartement des barreaux empêcher les animaux de l&#39;apprentissage de la localisation absolue et relative des barreaux et ainsi minimiser la capacité des animaux pour compenser les déficiences grâce à l&#39;apprentissage. De plus, en changeant l&#39;espacement entre les barreaux permet le test pour être utilisé à plusieurs reprises dans des études à long terme. Les méthodes sont décrites pour la description à la fois quantitative et qualitative des performances à la fois antérieurs et postérieurs, y compris des membres mise en place, pas à pas, de la coordination. Par ailleurs, l&#39;utilisation de stratégies compensatoires est indiqué par faux-pas ou des mesures compensatoires en réponse à un autre membre de malposition.

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.

Le barreau d&#39;échelle marcher tâche est un nouveau test pour évaluer la marche qualifiée et mesurer à la fois des membres antérieurs et postérieurs mise en place, pas à pas, et inter-membre de la coordination.

Le barreau d&#39;échelle Marche Tâche: Un système de notation et son application pratique.

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

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25.8K vues.  University of Lethbridge. Le barreau d'échelle marcher tâche est un nouveau test pour évaluer la marche qualifiée et mesurer à la fois des membres antérieurs et postérieurs mise en place, pas à pas, et inter-membre de la coordination.

Vidéo: 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;utilisation de Rotorod comme une méthode pour l&#39;analyse qualitative de la marche chez le 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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Biologie

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.

Objectif transplantation chez le poisson zèbre et son application dans l&#39;analyse de mutants 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.

Visualisation des Caenorhabditis elegans</emOuvrages cuticules> Utilisation du colorant lipophile 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.

Un Exonuclease analyse basée sur la fluorescence pour caractériser DmWRNexo, orthologue humain progéroïde WRN Exonuclease, et son application à d&#39;autres nucléases

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;

Un guide pratique pour Phylogenetics pour non-experts

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.

Conception et analyse du comportement de préférence de température et de son rythme circadien chez la drosophile

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.

Reconstruction de 3-Dimensional Volume histologique et son application à l&#39;étude de souris glandes mammaires

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.

Imagerie réorganisation spatiale d&#39;une voie de signalisation MAPK Utilisation du système d&#39;expression transitoire du tabac

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-Transformation génétique médiée, production transgénique et application pour l’étude du développement de la reproduction masculine dans le riz

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.