ここで紹介する研究はチャールズ川によってサポートされていました。

<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>

著者はチャールズ川の従業員です。

このプロトコルは、ハンズオン·研究者の施設で行う研修を補完するためのもの、動物取扱及び物質管理への導入として見られるべきである。使用され、物質の投与経路は、研究倫理委員会のプロトコルまたはプロトコルが書き込まれるときに実験的な設計で考慮しなければならない拘束する手段。 動物関連の手続きの訓練、研究の成功に不可欠です。ほとんどの実験を行うために、動物を研究スタッフによって処理される必要があり、より良い動物取扱、少ない動物3を強調した 。優しい人間との接触に動物を慣らし、ストレスを軽減し、より扱いやすい研究対象は4,5動物を作ることができます。ハンドリングストレスが研究6のいくつかの種類に影響を与えることが示され、それはそれは同様に他の人に影響を与えることが可能ですされています。げっ歯類の拘束を一時的な（慎重に、しかし、しっかりとしたハンドリングに達成されるべきであるグリップは、げっ歯類やハンドラへの傷害を招く可能性がある）と実用的な最短の期間中でなければなりません。拘束メソッドは、通常、動物の大きさに基づいて選ばれた、またはアクセスが求められている。例えば、可能なものの、首筋によって成体ラットを取り扱う、しばしばハンドラが未熟である場合は特に、ラットによる強い抵抗に会っている。手でマウスまたはラットを保有していることで尾静脈へのアクセスを困難にすることができると拘束装置は、多くの場合、まだ可能な限り動物を維持するために選択されます。 研究者は、動物を取り扱う際には、彼らはしばしば、さらなる研究のための化合物、または生物学的に管理しようとしている。物質の投与経路、吸収、生物学的利用能、および特定の実験のための適合性に影響を与えることができる。様々な経路を熟知自分の研究のために可能な限り最善の方法で彼らの物質を投与する能力を研究者に提供する必要があります。物質の急速な吸収を促進たとえば、ルート研究者はゆっくりと作用する方法で物質を投与しようとする場合、例えば、静脈内または腹腔内など、使用すべきではありません。ボリューム、機器、および溶質のため、これらの技術と問題のいくつかの最近のレビューは、ターナーらによる二つの論文で見つけられるかもしれません。1,7 物質は実験用げっ歯類に投与されるたびに、配慮が機器や物質の体積の適切なサイズ（ 表1に記載）に与えられるべきである。不適切なサイズの機器や、大量のは、不快感、傷害、または動物の死に至ることがある。一般的に、非経口的に投与する物質が研究の目標（ すなわち 、細菌の研究）は、これが不可能になる場合を除いて、滅菌されている。化合物と生物学的製剤は、動物に少なくとも影響を及ぼします溶質または車両でなければなりません。生理的pH（7.3 -7.4）は特に皮下、筋肉内およびintrapeため、一般的によく受け入れられているritonealルート。これらの経路によって投与物質の非生理的なpHレベルは、痛みや壊死や組織の損傷を引き起こす可能性があります。 pHの広い範囲は、胃内および静脈内経路7で許容される。小型げっ歯類では、別の重要な考慮事項は、室温での液体の大ボリュームが与えられている場合に冷えるの可能性である。流体は特に病気の動物を支援するために、静脈内または腹腔内投与されている場合、それらは体温（37℃）に温めなければならない。 このプロトコルで議論の投与経路は、マスターに単純であり、一般的には麻酔を必要としない、一般的に多くの研究プログラムで使用されているものです。投与経路のほとんど無限の多様性は、頭蓋内、髄腔内、硬膜外、気管内、骨内、関節内へと名前が、いくつかを含めて、しかし、可能です。政権のこれらの特殊なルートでのトレーニングは、人WHを求めるべきであるoはルートと良い結果と豊富な経験を持っています。 気管内注入より法、げっ歯類では、鼻腔内経路は、通常、より &quot;自然&quot;を介して肺に導入された物質を研究するために使用されています。マウスとラットは絶対的鼻呼吸であるため、流体の非常に少量を吸い込むためにそれらを誘導さえ意識のある動物では、難しいことではありません。鼻粘膜が血管によく供給されているので、いくつかの物質の鼻腔内投与、静脈内投与と同様とすることができる。このルートはその吸収を損なう可能性がありますので、しかし、鼻炎を持つ動物ではお勧めできません。鼻腔内経路によって大きなボリュームを管理しようとする試みは、動物の呼吸困難や溺死になることがあります。 筋肉内注射は物質の急速な吸収を提供します。筋肉内注射は、その小さなサイズとそれに対応して小さな筋肉に起因するラットやマウスに挑戦することができます。彼らは後ろ足で実行されsである。坐骨神経の損傷が発生する可能性があるため、大腿四頭筋は、選択肢の筋肉です。 皮下および皮内経路の両方が肌を伴うものの、皮下組織対皮膚に置か物質の生物学的利用率には違いがあります。皮下投与は、多くの場合、静脈内または腹腔内のような他のルートより遅い吸収と、 &quot;堆積&quot;ルートと見なされます。皮内投与は、通常、物質、そのようなアジュバントと抗原の混合物などの免疫賦活物質の非常に少量のために使用されます。両方のケースにおいて、投与物質は生理学的pHと非刺激性であるべきである。これはげっ歯類を抑制するための一般的に使用されるサイトですので、皮内または皮下注射は、首筋に行われるべきではない。 静脈内および腹腔内投与は、しばしば、げっ歯類に相当すると考えられている。以上のrを提供投与の静脈内経路化合物の取り込みAPIDしかし、腹腔内投与は、経口投与8とほぼ同等とみなされるべきである一方。不適切にバッファリングされた場合、彼らは痛みを引き起こす可能性があるため注意が化​​合物を腹腔内投与して撮影する必要があります。げっ歯類における静脈内ボーラス投与の一般的なルートは、尾静脈を介して行われます。物質の慢性静脈内投与が望ましい場合には、静脈または動脈カニューレの移植を考慮すべきである。静脈内に投与した物質は無菌的に配信されるべきであり、静脈内投与することが安全であることが示されるべきである。例えば、溶血、血栓症、または血管炎を誘発する可能性がある物質は、静脈内投与には適していません。 胃内または経口強制ルートはしばしばヒトで共通の投与経路を模倣するために使用されています。食べ物や水を介して経口投与と比較した場合、それはまた、物質の正確な投与を可能にします。化合物のバイオアベイラビリティ強制経口投与を介して投与した動物の飼育/絶食状態、ならびに化合物又は生物の溶質または車両によって異なります。強制経口投与または給餌針が使用されている動物に適したサイズである必要があり、使い捨て強制飼養針が実用的でない場合は、動物との間にクリーニングする必要があります。強制経口投与による傷害は珍しいものではなく、肺や胃や食道破裂への物質の堆積を含む。目を覚まし動物に強制経口投与が試行される前に、訓練は、経験豊富なパーティが監修して、最初の安楽死させた動物は、麻酔した動物（安楽死されるもの）で行われるべきである。目を覚ました動物の最初の強制経口投与の試みは手続きがゆがんで行く場合、傷害を引き起こすことはありません生理食塩水などの平均的なサイズの動物や物質の少量を、関与させるべきである。動物は密接に強制経口投与し、安楽死させた後、必要に応じて、青、出血、または過剰な唾液分泌を回すと、そのようなあえぎとして苦痛の徴候を評価すべきである。もしeuthanasiaが必要な場合は、動物は強制経口投与手順が失敗した理由を判断するために剖検されるべきである。

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td>針</td><td>さまざまな</td><td>さまざまな</td><td>針はゲージと長さの両方で販売されています。ご注文の前に両方をチェックしてください。 </td></tr><tr><td>シリンジ</td><td>さまざまな</td><td>さまざまな</td><td>常に投与すべきボリュームの適切なサイズを選択します。 </td></tr><tr><td> DecapiCones </td><td>トリーサイエン</td><td> DC-200は、DCL-120、MDC-200 </td><td>マウスおよびラットのサイズで利用できる。 </td></tr><tr><td>げっ歯類の制止</td><td>ハーバード装置、トリー·サイエンティフィック、プラス·ラボ、他</td><td></td><td>明確なプレキシガラス、調節可能なプラスチック、マウスおよびラットのためのサイズで利用できる。 </td></tr><tr><td> 50 mlコニカルチューブ</td><td>さまざまな</td><td> ＆nbのSPの; </td><td></td></tr><tr><td>餌針</td><td> VWR、ポッパーと息子</td><td>さまざまな</td><td>上記のような動物に針ゲージと長さを合わせます。両方の使い捨てと再利用可能な餌針が用意されています。 </td></tr></tbody></table>

manual restraint and common compound administration routes in mice and rats

安全かつ効果的にマウスやラットを抑制することができれば、研究を行うことの重要な部分です。マウスやラットで安心と人道的に働くことはハンドリングと拘束方法の基本的な能力を必要とします。この記事では、安全に動物を処理するために必要な基本的な原則を提示します。片手、両手、そして特別に設計された拘束オブジェクトと拘束を説明する。多くの場合、動物の研究やテスト使用の別の部分がマウスとラットへの化合物の効果的な投与である。可能な投与経路の数が多い（唯一の動物の大きさや臓器によって制限される）がありますが、ほとんどが研究に定期的に使用されていません。このビデオでは、静脈内、筋肉内、皮下、経口胃管栄養を含むより一般的な経路のいくつかを説明します。この記事の目的は、基本的な拘束や物質投与ルートにこれらの技術に不慣れなビューアを公開することです。このビデオあなたの施設で訓練に参加の必須に代わるものではありませんが、その訓練を補強し、補完することを意図している。

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

研究用げっ歯類で安全かつ人道的に働くことはハンドリングと拘束方法のコアコンピテンシーを必要とします。この記事では、安全に処理するために必要な基本的な原則を提示し、効果的にマウスやラットに化合物を投与するであろう。

マウスとラットの拘束マニュアルと共通の複合投与経路

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

manual-restraint-common-compound-administration-routes-mice

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Biology

Unit 1

General Pharmacological Principles

SCIENTISTS IN ACTION

288.9K ビュー.  Charles River. 研究用げっ歯類で安全かつ人道的に働くことはハンドリングと拘束方法のコアコンピテンシーを必要とします。この記事では、安全に処理するために必要な基本的な原則を提示し、効果的にマウスやラットに化合物を投与するであろう。

ビデオ: 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.

ラットにおける乳腺の形態と乳癌リスクの変化

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 ...

生物学

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.

診断剖検及びラット及びマウスで選択された組織とサンプル集

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.

ラットとマウスにおける卵巣摘出および17β-エストラジオールの交換：ビジュアルデモ

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

ライブの検出<em&gt;エシェリヒア·コリ</em&gt; O157：PMA-qPCRによりH7細胞

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. ...

Construction of an Affordable and Easy-to-Build Zebrafish Facility

In vivo biomedical research is pivotal to translate in vitro findings into clinical advances. Small academic institutions with limited resources find it virtually impossible to build and maintain typical rodent facilities for research. Zebrafish research has been demonstrated to be a valuable alternative for in vivo research in pharmacology, physiology, development and genetic studies. This article demonstrates that a functional zebrafish facility can be built in an easy and affordable manner. We demonstrate that such a facility could be built in about one working day with minimal tools and expertise. The cost of the 27 1.8 L fish tank zebrafish facility constructed in this study was approximately $1,500. We estimate that the maintenance of an initial working 150 fish colony for 3 months is $1,000. This project involved students, who were introduced to aquaculturing of zebrafish for research proposes.

A Zebrafish facility suitable for breeding and in vivo research is built in an easy and affordable manner. Maintenance is minimal. This facility is ideal for student involvement and independent research.

手ごろな価格で簡単に構築ゼブラフィッシュ施設の建設

In vivo biomedical research is pivotal to translate in vitro findings into clinical advances. Small academic institutions with limited resources find it ...

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.

植物プロモーター解析: 同定とルート、インゲンの根粒特異的プロモーターの解析

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.

思春期の発症を確認マウスおよびラットの不妊治療法を実行することで生殖能力の決定

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.

動物性食品中の サルモネラ菌 をスクリーニングし、培養分離から サルモネラ菌 を確認するためのループ媒介等温増幅

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.

重み付け相関ネットワークに基づく異なる生息地における根内微生物叢の発散

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.

手動受粉、顕微鏡検査、および S遺伝子型分析を使用した柑橘類の自己(非)適合性および相互(非)適合性関係の決定

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