La ricerca qui presentata è stata sostenuta da 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>

Gli autori sono dipendenti di Charles River.

Questo protocollo dovrebbe essere considerata come una introduzione alla movimentazione degli animali e somministrazione della sostanza lo scopo di completare una formazione pratica fornito presso lo stabilimento del ricercatore. I mezzi di contenzione che saranno utilizzati e le vie di somministrazione della sostanza dovrebbero essere considerati nel disegno sperimentale e quando il protocollo di ricerca o del comitato etico il protocollo è scritto. La formazione in procedure connesse agli animali è vitale per il successo della ricerca. Per eseguire la maggior parte degli esperimenti, gli animali devono essere trattati da personale di ricerca, e il migliore è il trattamento degli animali, tanto meno ha sottolineato l&#39;animale 3. Abituando gli animali per gentile contatto umano può ridurre lo stress e rendere gli animali soggetti di ricerca più trattabili 4,5. Gestione dello stress ha mostrato di influenzare alcuni tipi di ricerca 6 ed è possibile che può influenzare altri. Sistema di ritenuta dei roditori deve essere effettuata con attenzione, ma la gestione dell&#39;impresa (un tentativopresa può causare lesioni a roditori e gestore) e dovrebbe essere per il minor tempo pratico. Metodi di ritenuta di solito sono scelti in base alle dimensioni dell&#39;animale o la richiesta di accesso. Per esempio, la movimentazione ratti adulti per la nuca, anche se possibile, è spesso incontrato con forte resistenza dal ratto, soprattutto se il gestore è inesperto. In possesso di un topo o ratto a mano può rendere l&#39;accesso alle vene coda difficile e di un dispositivo di ritenuta è spesso scelto di mantenere l&#39;animale più fermo possibile. Quando i ricercatori di gestire gli animali, sono spesso cercano di amministrare un composto o biologico per ulteriori studi. La via di somministrazione di sostanze può influenzare l&#39;assorbimento, la biodisponibilità e idoneità per un particolare esperimento. La familiarità con vari percorsi devono fornire ai ricercatori la possibilità di amministrare la loro sostanza nel miglior modo possibile, per le loro ricerche. Per esempio, un percorso che promuove assorbimento rapido di una sostanza, Ad esempio endovenosa o intraperitoneale, non deve essere utilizzato se il ricercatore vuole somministrare il farmaco in un modo più lento effetto. Recensioni recenti di alcune di queste tecniche e considerazioni per il volume, attrezzature, e soluto si può trovare in due articoli da Turner et al. 1,7 Quando le sostanze devono essere somministrate per roditori da laboratorio, occorre tenere in considerazione la dimensione corretta del materiale e volume di sostanza (indicato in tabella 1). Non correttamente attrezzature di altra dimensione o grandi quantità può provocare disagio, lesioni o morte dell&#39;animale. In generale, le sostanze somministrate per via parenterale sono sterili, se gli obiettivi di ricerca renderebbe ciò impossibile (ad esempio, gli studi batteriche). Composti biologici e dovrebbe essere in un soluto o veicolo che avrà il minimo effetto sull&#39;animale. A pH fisiologico (7,3 -7,4) è generalmente ben accettato, soprattutto per via sottocutanea, intramuscolare, e intrapepercorsi ritoneal. Non fisiologici livelli di pH in sostanze somministrate attraverso queste vie può causare dolore o necrosi e danni ai tessuti. Più ampie gamme di pH sono tollerati con percorsi intragastrici ed endovenosa 7. In piccoli roditori, un&#39;altra considerazione importante è la possibilità di rilassarsi, se grandi quantità di liquidi a temperatura ambiente sono dati. Se i liquidi sono stati somministrati per via endovenosa o intraperitoneale, specialmente a sostegno di un animale malato, che deve essere riscaldato a temperatura corporea (37 ° C). Le vie di somministrazione discussi in questo protocollo sono quelli comunemente utilizzati in molti programmi di ricerca, sono semplici da padroneggiare, e generalmente non richiede anestesia. Una varietà quasi infinita di vie di somministrazione sono possibili, tuttavia, compreso intracranica, intratecale, epidurale, intratracheale, intraossea, e intra-solo per citarne alcuni. Formazione in questi percorsi specializzati di somministrazione dovrebbe essere cercato da persone who hanno una vasta esperienza con il percorso e buoni risultati. Nei roditori, la via intranasale è in genere utilizzato per studiare le sostanze introdotte per i polmoni attraverso una più &quot;naturale&quot;, metodo di instillazione. Topi e ratti sono obbligati naso-respiratori, così inducendoli a inalare piccole quantità di liquido non è difficile, anche in animali coscienti. Poiché la mucosa nasale è ben fornito con vasi sanguigni, la somministrazione intranasale di alcune sostanze può essere simile alla somministrazione endovenosa. Questo percorso è sconsigliato in animali con rinite, tuttavia, come che possono compromettere l&#39;assorbimento. I tentativi di amministrare grandi volumi per via endonasale può causare dispnea o annegamento dell&#39;animale. Iniezioni intramuscolari di fornire un rapido assorbimento delle sostanze. Iniezioni intramuscolari può essere difficile in ratti e topi a causa delle loro piccole dimensioni e di conseguenza i muscoli piccoli. Sono eseguiti nella zampa posteriores. A causa del rischio di danni del nervo sciatico, il quadricipite femorale è il muscolo della scelta. Sebbene sia il sottocutanea e intradermica coinvolgono la pelle, ci sono differenze tra la biodisponibilità di sostanze immesse nella pelle contro il sottocute. La somministrazione sottocutanea è spesso considerato un percorso &quot;deposizione&quot;, con più lento assorbimento di altre vie, ad esempio per via endovenosa o intraperitoneale. Somministrazione intradermica viene utilizzato per volumi molto piccoli di sostanze, tipicamente sostanze immunostimolanti come adiuvante-antigene miscele. In entrambi i casi, le sostanze somministrate devono essere di pH fisiologico e non irritante. Intradermica o sottocutanea iniezioni non deve essere eseguita nel nuca, poiché questo è un sito comunemente usato per trattenere il roditore. Somministrazione endovenosa e intraperitoneale sono spesso considerati equivalenti nei roditori. Endovenosa di dosaggio fornire maggiori rApID assorbimento di composti, tuttavia, mentre somministrazione intraperitoneale deve essere considerata approssimativamente equivalente a somministrazione orale 8. Si deve prestare attenzione per via intraperitoneale con composti somministrati in quanto possono causare dolore se impropriamente tamponata. Il percorso comune di somministrazione in bolo per via endovenosa nei roditori è attraverso le vene della coda. Se cronica somministrazione endovenosa di una sostanza è desiderato, l&#39;impianto di venosa o arteriosa cannule devono essere considerati. Le sostanze somministrate per via endovenosa deve essere consegnato in modo asettico e deve essere dimostrato di essere sicuro per somministrare per via endovenosa. Ad esempio, le sostanze che possono indurre emolisi, trombosi, vasculite o non sono adatte per la somministrazione per via endovenosa. Il percorso gavage intragastrico o orale è spesso utilizzato per simulare un percorso comune somministrazione nell&#39;uomo. Ciò consente inoltre di dosaggio preciso di sostanze rispetto alla somministrazione per via orale attraverso il cibo o acqua. Biodisponibilità dei compostisomministrato tramite gavage varierà in base allo stato alimentato / digiuno dell&#39;animale, nonché il soluto o veicolo del composto o biologiche. Aghi sonda gastrica o con l&#39;alimentazione devono essere di dimensioni adeguate per gli animali in uso, e deve essere pulita tra gli animali, se gli aghi monouso sonda gastrica non sono pratici. Infortuni causati da sonda gastrica non sono rari e includono la deposizione della sostanza nei polmoni o rottura dello stomaco o dell&#39;esofago. La formazione deve essere controllata da un soggetto esperto e intrapresi sugli animali eutanasia, poi animali anestetizzati (che saranno sacrificati) prima sonda gastrica su animali svegli è tentato. Primi tentativi di gavage sugli animali svegli dovrebbe coinvolgere medie dimensioni animali e piccoli volumi di una sostanza, come soluzione salina, che non provocano lesioni se la procedura va storto. Gli animali devono essere attentamente valutati per segni di sofferenza, come senza fiato, diventando blu, sanguinamento, o salivazione eccessiva, dopo sonda gastrica e eutanasia, se necessario. Se euthanasia è necessaria, l&#39;animale deve essere sottoposti a necroscopia per determinare perché la procedura di sonda gastrica non è riuscita.

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Aghi </td><td> Vario </td><td> Vario </td><td> Gli aghi sono venduti sia da calibro e la lunghezza. Controllare sia prima di ordinare. </td></tr><tr><td> Siringhe </td><td> Vario </td><td> Vario </td><td> Sempre scegliere una dimensione appropriata per il volume da somministrare. </td></tr><tr><td> DecapiCones </td><td> Braintree scientifico </td><td> DC-200, DCL-120, MDC-200 </td><td> Disponibile in topo e ratto dimensioni. </td></tr><tr><td> Roditori restrainer </td><td> Harvard Apparatus, Braintree scientifico, Plas-Labs, altri </td><td></td><td> Disponibile in plexiglas trasparente, in plastica regolabile, e dimensionati per topi e ratti. </td></tr><tr><td> Provetta da 50 ml </td><td> Vario </td><td> &amp; Nbsp; </td><td></td></tr><tr><td> Aghi di alimentazione </td><td> VWR, Popper and Sons </td><td> Vario </td><td> Montare l&#39;ago calibro e la lunghezza per gli animali come descritto sopra. Entrambi gli aghi di alimentazione monouso e riutilizzabili sono disponibili. </td></tr></tbody></table>

manual restraint and common compound administration routes in mice and rats

Essere in grado di trattenere in modo sicuro ed efficace topi e ratti è una parte importante di condurre ricerca. Lavorare con fiducia e umanità con i topi e ratti richiede una competenza di base in metodi di gestione e di moderazione. Questo articolo presenta i principi fondamentali necessari per gestire in modo sicuro gli animali. Con una sola mano, a due mani, e della moderazione con oggetti di ritenuta appositamente progettati saranno illustrati. Spesso, un&#39;altra parte della ricerca o sperimentazione di animali è la gestione efficace dei composti di topi e ratti. Anche se ci sono un grande numero di possibili vie di somministrazione (limitato solo dalle dimensioni e organi dell&#39;animale), la maggior parte non sono usati regolarmente nella ricerca. Questo video illustrerà più delle rotte più comuni, endovenosa, intramuscolare, sottocutanea, e sonda gastrica. L&#39;obiettivo di questo articolo è quello di esporre un visualizzatore di familiarità con queste tecniche di contenimento di base e vie di somministrazione della sostanza. Questo videonon sostituisce hands-on di richieste di formazione presso la vostra struttura, ma ha lo scopo di aumentare e completare tale formazione.

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

Lavorare in sicurezza ed umanità con roditori di ricerca richiede una competenza di base nei metodi di gestione e di moderazione. Questo articolo presenta i principi fondamentali necessari per gestire in modo sicuro ed efficace somministrare composti a topi e ratti.

Contenzione manuale e comuni Itinerari Compound amministrazione in topi e ratti

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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Pharmacology

Biology

Unit 1

General Pharmacological Principles

SCIENTISTS IN ACTION

288.0K Views.  Charles River. Lavorare in sicurezza ed umanità con roditori di ricerca richiede una competenza di base nei metodi di gestione e di moderazione. Questo articolo presenta i principi fondamentali necessari per gestire in modo sicuro ed efficace somministrare composti a topi e ratti.

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.

Cambiamenti nella morfologia della ghiandola mammaria e rischio di cancro al seno in topi

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 ...

Ricerca

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.

Necroscopia diagnostica e selezionati Collezione di tessuti e campioni di ratti e topi

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.

Sostituzione ovariectomia e 17β-estradiolo in ratti e topi: A dimostrazione visiva

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

Rilevamento dal vivo<em&gt; Escherichia coli</em&gt; O157: H7 Celle di 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.

Analisi del promotore di pianta: Identificazione e caratterizzazione di radice nodulo specifico promotore nel fagiolo comune

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.

Determinazione della competenza riproduttiva confermando Inizio puberale e realizzazione di un test di fertilità in topi e ratti

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.

Amplificazione isotermica mediata ad anello per lo screening della Salmonella negli alimenti animali e conferma della Salmonella dall'isolamento della coltura

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.

Divergenza del microbiota radicale in diversi habitat in base a reti di correlazione ponderate

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.

Determinazione delle relazioni di auto-(in)compatibilità e inter-(in)compatibilità negli agrumi mediante impollinazione manuale, microscopia e analisi del 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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Sviluppo di metodi di trasformazione e modifica genetica nel bambù

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.

Autore in primo piano: Avanzamento dell'editing genetico nelle foglie di bambù per alternative sostenibili alla plastica 

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.

A Planta Espressione genica e editing genico nelle foglie di bambù 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 ...