A pesquisa apresentada aqui foi apoiado 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. 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Os autores são funcionários da Charles River.

Este protocolo deve ser visto como uma introdução ao manejo dos animais e administração substância destina a complementar o treinamento prático desde a instalação do pesquisador. Os meios de contenção que será utilizado e as vias de administração substância deve ser considerada no planejamento experimental e quando o protocolo de pesquisa ou da comissão de ética está escrito. Treinamento em procedimentos relacionados a animais é vital para o sucesso da pesquisa. Para executar a maioria dos experimentos, os animais devem ser manuseados por pessoal de investigação, eo melhor manuseio do animal, a menos estressado o animal 3. Acostumar os animais ao contato humano gentil pode reduzir o estresse e fazer os animais sujeitos de pesquisa mais tratáveis ​​4,5. Estresse manuseamento tem sido demonstrado que afecta alguns tipos de pesquisa e 6, é possível que possa afectar outros. Contenção de roedores deve ser feito com cuidado, mas a manipulação de empresa (uma tentativaaderência é provável que resulte em prejuízo para roedores e manipulador) e deve ser para o menor período de tempo prático. Métodos de retenção são geralmente escolhidos com base no tamanho do animal ou o acesso requerido. Por exemplo, a manipulação ratos adultos pela nuca, embora possível, é muitas vezes encontrou-se com forte resistência dos ratos, especialmente se o manipulador é inexperiente. Segurando um rato ou o rato com a mão pode fazer o acesso às veias da cauda difícil e um dispositivo de retenção é geralmente escolhido para manter o animal como ainda possível. Quando os investigadores tratar animais, eles são muitas vezes pretende administrar um composto biológico ou para estudo posterior. A via de administração das substâncias podem afectar a absorção, biodisponibilidade, e uma aptidão para a experiência em particular. Familiaridade com várias rotas devem fornecer aos pesquisadores a capacidade de administrar os seus bens da melhor maneira possível para a sua pesquisa. Por exemplo, um percurso que promove a rápida absorção de uma substância, Tal como por via intravenosa ou intraperitoneal, não deve ser utilizado se o pesquisador deseja administrar a substância de uma forma mais lenta prolongada. Revisões recentes de algumas destas técnicas e considerações de volume, equipamento, e de solutos pode ser encontrada em dois artigos por Turner et al. 1,7 Sempre que as substâncias devem ser administradas a roedores de laboratório, deve considerar-se que o tamanho adequado do equipamento e o volume de substância (descritas no Quadro 1). Indevidamente equipamento de tamanho ou de grandes volumes pode resultar em desconforto, lesão ou morte do animal. Geralmente, substâncias administradas por via parentérica são estéreis, salvo os objetivos da pesquisa tornaria isso impossível (ou seja, estudos de bactérias). Compostos biológicos e deve estar em um soluto ou veículo que terá o menor efeito sobre o animal. Um pH fisiológico (7,3 -7,4) é geralmente bem aceite, especialmente para administração subcutânea, intramuscular, e intraperitoneal rotas. Níveis não fisiológicos de pH em substâncias administradas por estas vias pode resultar em dor ou necrose e danos nos tecidos. Faixas mais amplas de pH são tolerados com rotas intragástrica e intravenosa 7. Em roedores, uma outra consideração importante é a possibilidade de refrigeração, se grandes volumes de fluidos a temperatura ambiente são dados. Se os fluidos estão a ser administrados por via intravenosa ou intraperitoneal, em especial no apoio de um animal doente, que deve ser aquecida à temperatura corporal (37 ° C). As vias de administração discutidos neste protocolo são os mais utilizados em muitos programas de pesquisa, são simples de dominar, e geralmente não requer anestesia. Uma quase infinita variedade de vias de administração é possível, no entanto, incluindo intracraniana, intratecal, epidural, intratraqueal, intra-e intra-articular, para citar apenas alguns. Formação nestas rotas especializadas de administração deve ser procurado por pessoas who tem uma vasta experiência com a rota e bons resultados. Em roedores, a via intranasal é geralmente utilizada para estudar as substâncias introduzidas para os pulmões por meio de uma forma mais &quot;natural&quot;; método de instilação intratraqueal. Os ratinhos e ratos são obrigatórios nariz respiradores, assim induzindo-os a inalar quantidades muito pequenas de fluido não é difícil, até mesmo em animais conscientes. Uma vez que a mucosa nasal é bem suprida de vasos sanguíneos, a administração intranasal de algumas substâncias podem ser semelhantes aos da administração intravenosa. Esta via não é recomendado em animais com rinite, no entanto, uma vez que podem comprometer a absorção. As tentativas para administrar grandes volumes por via intranasal poderá resultar em dispneia ou afogamento do animal. Injecções intramusculares fornecer uma absorção rápida de substâncias. As injeções intramusculares pode ser um desafio em ratos e camundongos, devido ao seu pequeno tamanho e músculos correspondentemente pequenas. Elas são realizadas na pata traseiras. Devido ao potencial de danos do nervo ciático, femoral é o músculo quadriceps de escolha. Embora tanto a subcutânea e intradérmica envolvem a pele, existem diferenças entre a biodisponibilidade de substâncias colocadas na pele contra o subcutâneo. A administração subcutânea é frequentemente considerado como um &quot;depósito&quot; percurso, com absorção mais lenta do que as outras vias, tais como por via intravenosa ou intraperitoneal. A administração intradérmica é usado para volumes muito pequenos de substâncias, normalmente substâncias imunoestimulantes, tais como adjuvante de antigénio-misturas. Em ambos os casos, as substâncias devem ser administradas de pH fisiológico e não irritante. Intradérmica ou subcutânea injecções não deve ser realizada na nuca, uma vez que este é um local de restrição de uso geral para o roedor. A administração intravenosa e intraperitoneal são muitas vezes consideradas equivalentes em roedores. Intravenosa de dosagem proporcionam mais rAPID absorção de compostos, no entanto, ao passo que a administração intraperitoneal deve ser considerado equivalente a administração oral 8. Cuidados devem ser tomados com os compostos por via intraperitoneal, pois podem causar dor se indevidamente de buffer. A via comum de administração de bolus intravenoso, em roedores é através das veias da cauda. Se a administração intravenosa de uma substância crónica é desejada, a implantação de cânulas venosa ou arterial deve ser considerada. Substâncias administradas por via intravenosa deverá ser entregue de forma asséptica e deve ser mostrado para ser seguro para administrar por via intravenosa. Por exemplo, as substâncias que podem induzir a hemólise, trombose, ou vasculite não são apropriados para a administração intravenosa. A via de administração por sonda intragástrica ou por via oral é muitas vezes usado para imitar um percurso de dosagem comum nos seres humanos. Além disso, permite a dosagem exacta de substâncias em comparação com a administração oral, através de alimentos ou da água. A biodisponibilidade de compostosadministrado por gavagem irá variar com base no estado alimentado / jejum do animal, assim como o soluto ou o veículo ou do composto biológico. Agulhas gavage ou a alimentação deve ser de tamanho adequado para o animal a ser utilizado, e deve ser limpo entre os animais, se as agulhas descartáveis ​​gavage não são práticos. Lesões provocadas por gavagem não são incomuns e incluem a deposição da substância no pulmão ou ruptura do estômago ou do esófago. O treinamento deve ser supervisionado por um grupo experiente e realizado em animais sacrificados primeiro, então os animais anestesiados (que serão sacrificados) antes de gavagem em animais acordados é tentada. Primeiras tentativas gavage em animais acordados devem envolver animais de tamanho médio e pequenos volumes de uma substância, como o soro fisiológico, que não irá causar ferimentos se o processo der errado. Os animais devem ser cuidadosamente avaliados para sinais de sofrimento, como ofegante, virando sangramento, azul ou excesso de salivação, após gavagem e sacrificados, se necessário. Se euthanasia for necessário, o animal deve ser necropsiados para determinar por que o procedimento de gavagem falhou.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Agulhas </td><td> Vário </td><td> Vário </td><td> As agulhas são vendidos por ambos calibre e comprimento. Verifique tanto antes de encomendar. </td></tr><tr><td> Seringas </td><td> Vário </td><td> Vário </td><td> Sempre escolher um tamanho adequado para o volume a ser administrado. </td></tr><tr><td> DecapiCones </td><td> Braintree Científica </td><td> DC-200, DCL-120, MDC-200 </td><td> Disponível em mouse e tamanhos de ratos. </td></tr><tr><td> Limitador de roedores </td><td> Harvard Apparatus, Braintree Científica, Plas-Labs, outros </td><td></td><td> Disponível em vidro acrílico, plástico ajustável, e dimensionados para camundongos e ratos. </td></tr><tr><td> 50 ml tubo cónico </td><td> Vário </td><td> &amp; Nbsp; </td><td></td></tr><tr><td> Agulhas alimentação </td><td> VWR, Popper e Filhos </td><td> Vário </td><td> Ajustar o calibre e comprimento da agulha para os animais, tal como descrito acima. Ambas as agulhas descartáveis ​​e reutilizáveis ​​de alimentação estão disponíveis. </td></tr></tbody></table>

manual restraint and common compound administration routes in mice and rats

Ser capaz de forma segura e eficaz conter camundongos e ratos é uma parte importante da realização de pesquisas. Trabalhando com confiança e de forma humana com camundongos e ratos requer uma competência básica em métodos de manipulação e contenção. Este artigo irá apresentar os princípios básicos necessários para lidar com segurança com animais. Uma mão, de duas mãos, contenção e com objetos de retenção especialmente concebidos será ilustrado. Muitas vezes, uma outra parte da investigação ou utilização de animais de teste é a administração eficaz dos compostos a ratinhos e ratos. Embora haja um grande número de vias de administração possíveis (limitado apenas pelo tamanho e órgãos do animal), a maioria não são usadas regularmente em pesquisa. Este vídeo irá ilustrar várias das vias mais comuns, incluindo intravenosa, intramuscular, subcutânea, e por gavagem oral. O objetivo deste artigo é expor um espectador familiarizado com estas técnicas para contenção básico e vias de administração de substâncias. Este vídeonão substitui as mãos sobre a formação exigidos em suas instalações, mas destina-se a aumentar e completar o treinamento.

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

Trabalhar com segurança e de forma humana com roedores de investigação exige uma competência central em métodos de manipulação e contenção. Este artigo irá apresentar os princípios básicos necessários para lidar com segurança e efetivamente administrar compostos para camundongos e ratos.

Restrição manual e rotas de Administração composto comum em camundongos e ratos

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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Unit 1

General Pharmacological Principles

SCIENTISTS IN ACTION

288.0K visualizações.  Charles River. Trabalhar com segurança e de forma humana com roedores de investigação exige uma competência central em métodos de manipulação e contenção. Este artigo irá apresentar os princípios básicos necessários para lidar com segurança e efetivamente administrar compostos para camundongos e ratos.

Vídeo: 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.

Alterações na morfologia da glândula mamária e risco de câncer de mama em ratos

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 ...

Biologia

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.

Necropsia de diagnóstico e coleta de tecido e amostra selecionada em ratos e camundongos

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.

Ovariectomia e 17β-estradiol substituição em ratos e camundongos: uma demonstração 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

Detecção 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.

Análise do promotor de planta: Identificação e caracterização de raiz nódulo promotor específico no feijão comum

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.

Determinação da competência reprodutiva, confirmando o início puberal e realizando um ensaio de fertilidade em camundongos e ratos

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.

Amplificação isotérmica mediada por loop para triagem de salmonela em alimentos animais e confirmando salmonela do isolamento 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.

Divergência de Microbiota Raiz em Diferentes Habitats baseados em Redes de Correlação Ponderada

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.

Determinação das relações de auto-(in)compatibilidade e (in)compatibilidade em citros utilizando polinização manual, microscopia e análises de genótipo 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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Desenvolvendo métodos de transformação e edição de genes em bambu

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 em destaque: Avançando na edição de genes em folhas de bambu para alternativas sustentáveis de 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.

Em Planta Expressão Gênica e Edição Gênica em Folhas de Bambu 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 ...