La investigación presentada aquí fue apoyada por Charles River.

<ol>
	<li>Turner, P. V., Brabb, T., Pekow, C., Vasbinder, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Administration+of+substances+to+laboratory+animals:+routes+of+administration+and+factors+to+consider.">Administration of substances to laboratory animals: routes of administration and factors to consider.</a> JAALAS. 50, 600-613 (2011).</li><li>Diehl, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+good+practice+guide+to+the+administration+of+substances+and+removal+of+blood,+including+routes+and+volumes.">A good practice guide to the administration of substances and removal of blood, including routes and volumes.</a> J. Appl. Toxicol. 21, 15-23 (2001).</li><li>Hurst, J. L., West, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taming+anxiety+in+laboratory+mice.">Taming anxiety in laboratory mice.</a> Nat. Methods. 7, 825-826 (2010).</li><li>Maurer, B. M., D&#246;ring, D., Scheipl, F., K&#252;chenhoff, H., Erhard, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+a+gentling+programme+on+the+behaviour+of+laboratory+rats+towards+humans.">Effects of a gentling programme on the behaviour of laboratory rats towards humans.</a> Appl. Anim. Behav. Sci. 111, 329-341 (2008).</li><li>Cloutier, S., Newberry, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+conditioning+technique+to+reduce+stress+associated+with+repeated+intra-peritoneal+injections+in+laboratory+rats.">Use of a conditioning technique to reduce stress associated with repeated intra-peritoneal injections in laboratory rats.</a> Appl. Anim. Behav. Sci. 112, 158-173 (2008).</li><li>Romanovsky, A. A., Kulchitsky, V. A., Simons, C. T., Sugimoto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodology+of+fever+research:+why+are+polyphasic+fevers+often+thought+to+be+biphasic.">Methodology of fever research: why are polyphasic fevers often thought to be biphasic.</a> Am. J. Physiol. 275, 332-338 (1998).</li><li>Turner, P. V., Pekow, C., Vasbinder, M. A., Brabb, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Administration+of+substances+to+laboratory+animals:+equipment+considerations,+vehicle+selection,+and+solute+preparation.">Administration of substances to laboratory animals: equipment considerations, vehicle selection, and solute preparation.</a> JAALAS. 50, 614-627 (2011).</li><li>Lukas, G., Brindle, S. D., Greengard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+route+of+absorption+of+intraperitoneally+administered+compounds.">The route of absorption of intraperitoneally administered compounds.</a> J. Pharmacol. Exp. Ther. 178, 562-564 (1971).</li><li>AALAS. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory Mouse Handbook. , (2009).</li><li>AALAS. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> LAT Training Manual. , (2009).</li><li>AALAS. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> LATg Training Manual. , (2009).</li><li>Barnett, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual of Animal Technology. , 440 (2007).</li><li>Baumans, V., Pekow, C. A., Hau, J., Schapiro, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Laboratory Animal Science. 1, 401-446 (2010).</li><li>Bogdanske, J. J., Hubbard-Van Stelle, S., Riley, M. R., Schiffman, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory Mouse Procedural Techniques. , (2011).</li><li>Danneman, P., Suckow, M. A., Brayton, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Laboratory Mouse. , (2000).</li><li>Sharp, P. E., La Regina, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Laboratory Rat. , (1998).</li></ol>

Los autores son empleados de Charles River.

Este protocolo debe ser visto como una introducción al manejo de los animales y la administración de sustancias destinadas a complementar la formación práctica prevista en las instalaciones del investigador. Los medios de sujeción que se utilizará y las vías de administración sustancia debe ser considerada en el diseño experimental y cuando el protocolo de investigación o comité de ética de protocolo está escrito. Formación en procedimientos relacionados con los animales es vital para el éxito de la investigación. Para llevar a cabo la mayoría de los experimentos, los animales serán manipulados por el personal de investigación, y el mejor es el manejo de los animales, menos hincapié en el animal 3. Acostumbrar a los animales al contacto humano suave puede reducir el estrés y hacer que los animales sujetos de investigación más manejables 4,5. Manejo de estrés se ha demostrado que afecta a algunos tipos de investigación 6 y es posible que puede afectar a los demás. Restricción de roedores debe realizarse con cuidado, pero la gestión de la empresa (una tentativaagarre es probable que resulte en daño a los roedores y el guía) y debe ser durante el menor tiempo práctico. Métodos de inmovilización se suelen elegir en función del tamaño del animal o buscó el acceso. Por ejemplo, el manejo de las ratas adultas por la piel, aunque es posible, a menudo se encontró con una fuerte resistencia de la rata, especialmente si el controlador no tiene experiencia. Sosteniendo un ratón o una rata a mano puede hacer que el acceso a las venas de la cola difícil y un dispositivo de sujeción se elige a menudo para mantener al animal lo más quieto posible. Cuando los investigadores manejar animales, a menudo se busca para administrar un compuesto o biológico para el estudio adicional. La vía de administración de las sustancias pueden afectar la absorción, la biodisponibilidad, y la idoneidad para un experimento particular. La familiaridad con varias rutas deben proporcionar a los investigadores con la capacidad de administrar sus bienes de la mejor manera posible para su investigación. Por ejemplo, una ruta que promueve la absorción rápida de una sustancia, Tal como intravenosa o intraperitoneal, no debe usarse si el investigador desea administrar la sustancia de una manera más lenta de acción prolongada. Revisiones recientes de algunas de estas técnicas y consideraciones para el volumen, el equipo, y el soluto se pueden encontrar en dos artículos por Turner et al. 1,7 Siempre que las sustancias se administran a roedores de laboratorio, se debe tener en cuenta el tamaño adecuado del equipo y el volumen de la sustancia (que se señala en la Tabla 1). Incorrectamente equipos de tamaño o grandes cantidades puede ocasionar molestias, lesiones o muerte del animal. En general, las sustancias administradas por vía parenteral son estériles, salvo que de los objetivos de la investigación que hagan imposible (es decir, estudios, bacterianas). Los compuestos y sustancias biológicas deben estar en un soluto o vehículo que tiene el menor efecto sobre el animal. A pH fisiológico (7,3 -7,4) es generalmente bien aceptado, especialmente para administración subcutánea, intramuscular, y intraperutas ritoneal. No fisiológicas los niveles de pH en sustancias administradas por estas rutas puede dar lugar a dolor o necrosis y daño tisular. Intervalos más amplios de pH se toleran con rutas intragástricas e intravenosa 7. En roedores pequeños, otra consideración importante es la posibilidad de refrigeración si grandes volúmenes de fluidos de temperatura ambiente se les da. Si los líquidos se administran por vía intravenosa o por vía intraperitoneal, especialmente en apoyo de un animal enfermo, que debe calentarse a la temperatura corporal (37 ° C). Las vías de administración descritos en este protocolo son los de uso común en muchos programas de investigación, son fáciles de dominar, y generalmente no requiere anestesia. Una casi infinita variedad de vías de administración son posibles, sin embargo, incluyendo intracraneal, intratecal, epidural, intratraqueal intraósea, intraarticular y para nombrar sólo unos pocos. La formación en estas rutas especializadas de la administración se debería pedir a las personas quo tienen una amplia experiencia con la ruta y buenos resultados. En roedores, la vía intranasal se utiliza típicamente para estudiar sustancias introducidas a los pulmones a través de una más &quot;natural&quot;; método de instilación intratraqueal. Los ratones y las ratas son obligados nariz-respiraderos, de modo induciéndolos a inhalar cantidades muy pequeñas de fluido no es difícil, incluso en animales conscientes. Desde la mucosa nasal está bien provisto de los vasos sanguíneos, la administración intranasal de algunas sustancias pueden ser similares a la administración intravenosa. Esta ruta no se recomienda en animales con rinitis, sin embargo, como que puede comprometer la absorción. Los intentos para administrar grandes volúmenes por vía intranasal puede causar disnea o ahogo del animal. Las inyecciones intramusculares proporcionar una rápida absorción de las sustancias. Las inyecciones intramusculares pueden ser un reto en ratas y ratones, debido a su pequeño tamaño y los músculos correspondientemente pequeñas. Se realizan en la pata traseras. Debido a la posibilidad de daño del nervio ciático, el cuádriceps femoral es el músculo de elección. Aunque tanto la forma subcutánea o intradérmica afectan a la piel, existen diferencias entre la biodisponibilidad de las sustancias colocadas en la piel frente a la hipodermis. La administración subcutánea a menudo se considera una &quot;deposición&quot; ruta, con una absorción más lenta que otras rutas, tales como intravenosa o intraperitoneal. La administración intradérmica se utiliza para volúmenes muy pequeños de sustancias, típicamente de sustancias inmunoestimulantes, tales como mezclas de antígeno-adyuvante. En ambos casos, las sustancias administradas deben ser de pH fisiológico y no irritante. Intradérmica o subcutánea no se debe realizar en la nuca, ya que esto es un sitio de restricción de uso general para el roedor. La administración intravenosa e intraperitoneal a menudo se consideran equivalentes en roedores. Las vías intravenosa de dosificación proporcionan más rcaptación apid de compuestos, sin embargo, mientras que la administración intraperitoneal debe ser considerado aproximadamente equivalente a la administración oral 8. Se debe tener cuidado con los compuestos administrados por vía intraperitoneal, ya que pueden causar dolor si no se tamponado. La ruta común de la administración de bolo intravenoso en roedores es a través de las venas de la cola. Si la administración crónica intravenosa de una sustancia se desea, la implantación de cánulas venosa o arterial debe ser considerado. Sustancias administradas por vía intravenosa debe ser entregado asépticamente y debe ser demostrado ser segura para administrar por vía intravenosa. Por ejemplo, las sustancias que pueden inducir la hemólisis, trombosis, vasculitis o no son apropiados para la administración intravenosa. La ruta sonda intragástrica u oral se utiliza a menudo para imitar una ruta de dosificación común en los seres humanos. También permite una dosificación precisa de las sustancias en comparación con la administración oral a través de los alimentos o el agua. La biodisponibilidad de los compuestosadministrado a través de sonda variará basado en el estado de alimentación / ayuno del animal, así como el soluto o vehículo del compuesto o biológico. Agujas por sonda o alimentación debe ser del tamaño apropiado para el animal que está siendo utilizado, y se deben limpiar entre los animales, si agujas desechables por sonda no son prácticos. Las lesiones causadas por sonda no son infrecuentes e incluyen la deposición de la sustancia en el pulmón o la rotura del estómago o el esófago. La capacitación debe ser supervisado por un partido experimentado y realizado en animales sacrificados en primer lugar, entonces los animales anestesiados (que serán sacrificados) antes sonda en animales despiertos se intenta. Primeros intentos por sonda nasogástrica en animales despiertos debe involucrar promedio animales de tamaño y volúmenes pequeños de una sustancia, tal como solución salina, que no causen daño si el procedimiento sale mal. Los animales deben ser cuidadosamente evaluados para detectar signos de angustia, como el jadeo, convirtiendo sangrado azul, o salivación excesiva, después de alimentación forzada y la eutanasia si es necesario. Si euthAnasia se requiere, el animal debe ser necropsia para determinar por qué el procedimiento de sonda falló.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Needles </td><td> Vario </td><td> Vario </td><td> Las agujas se venden por tanto calibre y longitud. Compruebe tanto antes de ordenar. </td></tr><tr><td> Jeringas </td><td> Vario </td><td> Vario </td><td> Siempre elegir un tamaño apropiado para el volumen a administrar. </td></tr><tr><td> DecapiCones </td><td> Braintree Scientific </td><td> DC-200, DCL-120, MDC-200 </td><td> Disponible en ratón y rata tamaños. </td></tr><tr><td> Roedores restrainer </td><td> Harvard Apparatus, Braintree Scientific, Plas-Labs, otros </td><td></td><td> Disponible en Plexiglas, de plástico ajustable, y un tamaño para ratones y ratas. </td></tr><tr><td> Tubo cónico de 50 ml </td><td> Vario </td><td> &amp; Nbsp; </td><td></td></tr><tr><td> Agujas de alimentación </td><td> VWR, Popper and Sons </td><td> Vario </td><td> Coloque el calibre y longitud de aguja a los animales como se describió anteriormente. Ambas agujas de alimentación desechables y reutilizables están disponibles. </td></tr></tbody></table>

manual restraint and common compound administration routes in mice and rats

Ser capaz de reprimir con seguridad y eficacia ratones y ratas es una parte importante de la realización de investigaciones. Trabajar con seguridad y humanamente con los ratones y las ratas requiere un conocimiento básico de los métodos de manipulación y sujeción. En este artículo se presentan los principios básicos necesarios para manejar con seguridad los animales. Con una sola mano, a dos manos, y la restricción de objetos de sujeción especialmente diseñados se ilustra. A menudo, otra parte de la investigación o el uso de pruebas de los animales es la administración efectiva de los compuestos a ratones y ratas. Aunque hay un gran número de posibles vías de administración (limitado sólo por el tamaño y órganos del animal), la mayoría no se utilizan regularmente en la investigación. Este vídeo se ilustran varias de las rutas más comunes, incluyendo la administración intravenosa, intramuscular, subcutánea, y una sonda oral. El objetivo de este artículo es exponer un espectador familiarizado con estas técnicas a la restricción básica y vías de administración de sustancias. Este videono reemplaza práctica requeridas capacitación en sus instalaciones, pero tiene la intención de aumentar y complementar la formación.

Watch this Scientific Journal Video about Manual Restraint and Common Compound Administration Routes in Mice and Rats at JoVE.com

Manual Restraint and Common Compound Administration Routes in Mice and Rats

Trabajar con seguridad y humanamente con los roedores de investigación requiere una competencia básica en métodos de manipulación y sujeción. En este artículo se presentan los principios básicos que se requieren para manejar con seguridad y eficacia administrar compuestos a ratones y ratas.

Sujeción manual y de las rutas de la administración del compuesto en ratones y ratas

Being able to safely and effectively restrain mice and rats is an important part of conducting research.  Working confidently and humanely with mice and ...

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Research

Education

JoVE Core

JoVE Journal

Pharmacology

Biology

Unit 1

General Pharmacological Principles

SCIENTISTS IN ACTION

288.0K vistas.  Charles River. Trabajar con seguridad y humanamente con los roedores de investigación requiere una competencia básica en métodos de manipulación y sujeción. En este artículo se presentan los principios básicos que se requieren para manejar con seguridad y eficacia administrar compuestos a ratones y ratas.

Video: Manual Restraint and Common Compound Administration Routes in Mice and Rats

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland undergoes extensive modeling and re-modeling, alter susceptibility to carcinogen-induced mammary tumors. Similar findings have been described in humans: for example, high birthweight increases later risk of developing breast cancer, and dietary intake of soy during childhood decreases breast cancer risk. It is thought that these prenatal and postnatal dietary modifications induce persistent morphological changes in the mammary gland that in turn modify breast cancer risk later in life. These morphological changes likely reflect epigenetic modifications, such as changes in DNA methylation, histones and miRNA expression that then affect gene transcription . In this article we describe how changes in mammary gland morphology can predict mammary cancer risk in rats. Our protocol specifically describes how to dissect and remove the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology according to three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use the data to predict risk of developing mammary cancer.

Our protocol describes how to dissect the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology using three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use these results to predict mammary cancer risk in rats which were exposed to dietary modifications in utero or during prepuberty.

Los cambios en la morfología de la glándula mamaria y el riesgo de cáncer de mama en ratas

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland ...

Investigación

Biología

Diagnostic Necropsy and Selected Tissue and Sample Collection in Rats and Mice

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal colony. Proper collection of tissues for further histological processing can impact the quality of testing results. This article describes the conduct of a basic gross examination including identification of heart, liver, lungs, kidneys, and spleen, as well as how to collect those organs. Additionally four of the more difficult tissue/sample collection techniques are demonstrated. Lung collection and perfusion can be particularly challenging as the tissue needs to be properly inflated with a fixative in order for inside of the tissue to fix properly and to enable thorough histologic evaluation. This article demonstrates the step by step technique to remove the lung and inflate it with fixative in order to achieve optimal fixation of the tissue within 24 hours. Brain collection can be similarly challenging as the tissue is soft and easily damaged. This article demonstrates the step by step technique to expose and remove the brain from the skull with minimal damage to the tissue. The mesenteric lymph node is a good sample type in which to detect many common infectious agents as enteric viruses persist longer in the lymph node than they are shed in feces. This article demonstrates the step by step procedure for locating and aseptically removing the mesenteric lymph node. Finally, identification of infectious agents of the respiratory tract may be performed by bacterial culture or PCR testing of nasal and/or bronchial fluid aspirates taken at necropsy. This procedure describes obtaining and preparing the respiratory aspirate sample for bacterial culture and PCR testing.

This article describes the procedures for conducting a basic postmortem examination of a mouse or rat, and the collection of basic organs, as well as more challenging sample types from for histological, microbiological, and PCR evaluation.

La necropsia de diagnóstico y selecta colección de tejidos y muestras en ratas y ratones

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal ...

Ovariectomy and 17&beta;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are commonly ovariectomized in order to ablate the rapidly cycling hormone production, replacing the 17&beta;-estradiol exogenously. There is, however, lack of consensus regarding how the hormone should be administered to obtain physiological serum concentrations. This is crucial since the 17&beta;-estradiol level/administration method profoundly influences the experimental results1-3. We have in a series of studies characterized the different modes of 17&beta;-estradiol administration, finding that subcutaneous silastic capsules and per-oral nut-cream Nutella are superior to commercially available slow-release pellets (produced by the company Innovative Research of America) and daily injections in terms of producing physiological serum concentrations of 17&beta;-estradiol4-6. Amongst the advantages of the nut-cream method, that previously has been used for buprenorphine administration7, is that when used for estrogen administration it resembles peroral hormone replacement therapy and is non-invasive. The subcutaneous silastic capsules are convenient and produce the most stable serum concentrations. This video article contains step-by-step demonstrations of ovariectomy and 17&beta;-estradiol hormone replacement by silastic capsules and peroral Nutella in rats and mice, followed by a discussion of important aspects of the administration procedures.

Ovariectomy and 17&amp;#946;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Ovariectomy and subsequent replacement of 17&beta;-estradiol by means of silastic capsules and peroral Nutella are demonstrated in rats and mice.

Ovariectomía y 17β-estradiol reemplazo en ratas y ratones: una demostración visual

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

La detección de vivo<em&gt; Escherichia coli</em&gt; O157: H7 células por PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in understanding the gene regulation and spatiotemporal expression patterns of target genes. On the other hand, it is also critical to establish promoter evaluation tools and genetic transformation techniques that are fast, efficient, and reproducible. In this study, we investigated the spatiotemporal expression pattern of the rhizobial symbiosis-specific nodule inception (NIN) promoter of Phaseolus vulgaris in the transgenic hairy roots. Using plant genome databases and analysis tools we identified, isolated, and cloned the P. vulgaris NIN promoter in a transcriptional fusion to the chimeric reporter &#946;-glucuronidase (GUS) GUS-enhanced::GFP. Further, this protocol describes a rapid and versatile system of genetic transformation in the P. vulgaris using Agrobacterium rhizogenes induced hairy roots. This system generates &#8805;2 cm hairy roots at 10 to 12 days after transformation. Next, we assessed the spatiotemporal expression of NIN promoter in Rhizobium inoculated hairy roots at periodic intervals of post-inoculation. Our results depicted by GUS activity show that the NIN promoter was active during the process of nodulation. Together, the present protocol demonstrates how to identify, isolate, clone, and characterize a plant promoter in the common bean hairy roots. Moreover, this protocol is easy to use in non-specialized laboratories.

Promoter expression analyses are crucial to improving the understanding of gene regulation and the spatiotemporal expression of target genes. Herein we present a protocol to identify, isolate, and clone a plant promoter. Further, we describe the characterization of the nodule-specific promoter in the common bean hairy roots.

Planta promotor análisis: Identificación y caracterización del promotor de la raíz específica de nódulo en el frijol común

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in ...

Determination of Reproductive Competence by Confirming Pubertal Onset and Performing a Fertility Assay in Mice and Rats

Assessment of reproductive competence is critical for understanding the impact of a treatment or genetic manipulation on the reproductive axis, also termed the hypothalamic-pituitary-gonadal axis. The reproductive axis is a key integrator of environmental and internal input adapting fertility to favorable conditions for reproduction. Prior to embarking upon a fertility study in mice and rats, sexual maturity is evaluated to exclude the possibility that the observed reproductive phenotypes are caused by delayed or absent pubertal onset. This protocol describes a non-invasive approach to assess pubertal onset in males through the determination of preputial separation, and in females through vaginal opening and first estrus. After the confirmation of the completion of puberty and the achievement of sexual maturity, a fertility study can be initiated. The procedure describes the optimal breeding conditions for mice and rats, how to set up a fertility study, and what parameters to evaluate and determine if the treatment or gene deletion has an impact on fertility.

Many treatments and genetic mutations impact the timing of sexual maturity and fertility. This protocol describes a non-invasive method to evaluate pubertal onset in mice and rats prior to setting up a fertility study in sexually mature animals.

Determinación de la capacidad reproductiva por confirmar Inicio puberal y de realizar un análisis de fertilidad en ratones y ratas

Assessment of reproductive competence is critical for understanding the impact of a treatment or genetic manipulation on the reproductive axis, also ...

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

Loop-mediated isothermal amplification (LAMP) has emerged as a powerful nucleic acid amplification test for the rapid detection of numerous bacterial, fungal, parasitic, and viral agents. Salmonella is a bacterial pathogen of worldwide food safety concern, including food for animals. Presented here is a multi-laboratory-validated Salmonella LAMP protocol that can be used to rapidly screen animal food for the presence of Salmonella contamination and can also be used to confirm presumptive Salmonella isolates recovered from all food categories. The LAMP assay specifically targets the Salmonella invasion gene (invA) and is rapid, sensitive, and highly specific. Template DNAs are prepared from enrichment broths of animal food or pure cultures of presumptive Salmonella isolates. The LAMP reagent mixture is prepared by combining an isothermal master mix, primers, DNA template, and water. The LAMP assay runs at a constant temperature of 65 &#176;C for 30 min. Positive results are monitored via real-time fluorescence and can be detected as early as 5 min. The LAMP assay exhibits high tolerance to inhibitors in animal food or culture medium, serving as a rapid, reliable, robust, cost-effective, and user-friendly method for screening and confirming Salmonella. The LAMP method has recently been incorporated into the U.S. Food and Drug Administration&#8217;s Bacteriological Analytical Manual (BAM) Chapter 5.

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification test (iNAAT) that has attracted broad interest in the pathogen detection field. Here, we present a multi-laboratory-validated Salmonella LAMP protocol as a rapid, reliable, and robust method for screening Salmonella in animal food and confirming presumptive Salmonella from culture isolation.

Amplificación isotérmica mediada por bucle para la detección de salmonela en alimentos para animales y la confirmación de salmonela del aislamiento cultural

Loop-mediated isothermal amplification (LAMP) has emerged as a powerful nucleic acid amplification test for the rapid detection of numerous bacterial, ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, which can effectively explore the interaction relationship or co-occurrence model of different microbial species in different environments. The purpose of this manuscript is to provide details on how to use the weighted correlation network algorithm to analyze different co-occurrence networks that may occur in microbial communities due to different ecological environments. All analysis of the experiment is performed in the WGCNA package. WGCNA is an R package for weighted correlation network analysis. The experimental data used to demonstrate these methods were microbial community data from the NCBI (National Center for Biotechnology Information) database for three niches of the rice (Oryza sativa) root system. We used the weighted correlation network algorithm to construct co-abundance networks of microbial community in each of the three niches. Then, differential co-abundance networks among endosphere, rhizoplane and rhizosphere soil were identified. In addition, the core genera in network were obtained by the "WGCNA" package, which plays an important regulated role in network functions. These methods enable researchers to analyze the response of microbial network to environmental disturbance and verify different microbial ecological response theories. The results of these methods show that the significant differential microbial networks identified in the endosphere, rhizoplane and rhizosphere soil of rice.

Network analysis was applied to evaluate the association of various ecological microbial communities, such as soil, water and rhizosphere. Presented here is a protocol on how to use the WGCNA algorithm to analyze different co-occurrence networks that may occur in the microbial communities due to different ecological environments.

Divergencia de la microbiota radicular en diferentes hábitats basada en redes de correlación ponderadas

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...

Determination of Self-(In)compatibility and Inter-(In)compatibility Relationships in Citrus Using Manual Pollination, Microscopy, and S-Genotype Analyses

Citrus uses S-RNase-based self-incompatibility to reject self-pollen and, therefore, requires nearby pollinizer trees for successful pollination and fertilization. However, identifying suitable varieties to serve as pollinizers is a time-consuming process. To solve this problem, we have developed a rapid method for identifying pollination-compatible citrus cultivars that utilizes agarose gel electrophoresis and aniline blue staining. Pollen compatibility is determined based on the identification of S genotypes by extracting total DNA and performing PCR-based genotyping assays with specific primers. Additionally, styles are collected 3-4 days after manual pollination, and aniline blue staining is performed. Finally, the growth status of the pollen tubes is observed with a fluorescence microscope. Pollen compatibility and incompatibility can be established by observing whether the pollen tube growth is normal or suppressed, respectively. Due to its simplicity and cost-effectiveness, this method is an effective tool for determining the pollen compatibility and incompatibility of different citrus varieties to establish incompatibility groups and incompatibility relationships between different cultivars. This method provides information essential for the successful selection of suitable pollinizer trees and, thus, facilitates the establishment of new orchards and the selection of appropriate parents for breeding programs.

Determination of Self-Incompatibility and Inter-Incompatibility Relationships in Citrus Using Manual Pollination, Microscopy, and S-Genotype Analyses

This protocol provides a rapid method for determining pollen compatibility and incompatibility in citrus cultivars.

Determinación de las relaciones de autocompatibilidad e intercompatibilidad en cítricos mediante polinización manual, microscopía y análisis de genotipo S

Citrus uses S-RNase-based self-incompatibility to reject self-pollen and, therefore, requires nearby pollinizer trees for successful pollination and ...

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Desarrollo de métodos de transformación y edición de genes en bambú

In Planta Gene Expression and Gene Editing in Moso Bamboo Leaves

A novel in planta gene transformation method was developed for bamboo, which avoids the need for time-consuming and labor-intensive callus induction and regeneration processes. This method involves Agrobacterium-mediated gene expression via wounding and vacuum for bamboo seedlings. It successfully demonstrated the expression of exogenous genes, such as the RUBY reporter and Cas9 gene, in bamboo leaves. The highest transformation efficiency for the accumulation of betalain in RUBY seedlings was achieved using the GV3101 strain, with a percentage of 85.2% after infection. Although the foreign DNA did not integrate into the bamboo genome, the method was efficient in expressing the exogenous genes. Furthermore, a gene editing system has also been developed with a native reporter using this method, from which an in situ mutant generated by the edited bamboo violaxanthin de-epoxidase gene (PeVDE) in bamboo leaves, with a mutation rate of 17.33%. The mutation of PeVDE resulted in decreased non-photochemical quenching (NPQ) values under high light, which can be accurately detected by a fluorometer. This makes the edited PeVDE a potential native reporter for both exogenous and endogenous genes in bamboo. With the reporter of PeVDE, a cinnamoyl-CoA reductase gene was successfully edited with a mutation rate of 8.3%. This operation avoids the process of tissue culture or callus induction, which is quick and efficient for expressing exogenous genes and endogenous gene editing in bamboo. This method can improve the efficiency of gene function verification and will help reveal the molecular mechanisms of key metabolic pathways in bamboo.

Autor destacado: Avance de la edición genética en hojas de bambú para alternativas sostenibles al plástico 

In this study, a novel in planta gene expression and gene editing method mediated by Agrobacterium was developed in bamboo. This method greatly improved the efficiency of gene function validation in bamboo, which has significant implications for accelerating the process of bamboo breeding.

En Planta Expresión génica y edición génica en hojas de bambú Moso

A novel in planta gene transformation method was developed for bamboo, which avoids the need for time-consuming and labor-intensive callus induction and ...