これらの研究は、DWLにCIHRとCBCRA補助金によって賄われた。 IPは、CIHR - STP、FRSQとCIHRから奨学金によって賄われていた。 MKGSはOGSとCIHR - STPの奨学金によって賄われていた。著者らは、マウスの繁殖との彼の援助のためにケビンバーに感謝。

1。乳腺の解剖<ol><li> CO 2吸入を使用して、マウスを安楽死させる。それは解剖がより困難にレンダリングする、乳腺の周りの血液の蓄積の首と、結果に大きな血管に損傷を与える可能性があるので、可能であれば、頸椎脱臼を避けてください。しかし、他の基準がそのようなオキシトシンの血中濃度として、同じマウスで評価される場合は、代替の安楽死の方法は、これはあなたのIACUCによって評価しなければならないのに、必要な場合があります。 </li><li>ホイルに包んで、発泡スチロールに、その背中の上でマウスを配置し、四肢に広がる。しっかりと16から20ゲージの針（図1A）を使用して、すべての四肢を固定する。 </li><li> 70％エタノールで寛大にマウスをスプレーしてください。 </li><li>鉗子を使用して、正中線で腹部の皮膚を引き上げ、鋭いはさみと小さな切開を行います。 </li><li>腹部や胸部の空洞（図1B）をパンクチャ回避しながら切開から始めて、動物の首に皮膚をカット。 </li><li> &quot;Yの形&quot;（図1B）、その結果、正中切開に前足で皮膚を切り取ります。リア手足の皮膚と同じ方法（図1B）をカット。 </li><li>静かにマウスの側面に腹部と胸部の皮膚剥離、止血鉗子を使用する。必要に応じて、穏やかに論理積組織をカット。 </li><li>皮膚の下側に接続されている乳腺を公開し、16〜20ゲージの針を使用して、発泡スチロールにスキンを修正。進むと時間（図1C）で片側を完了します。 </li><li>鉗子を使用して、静かに動物の背骨に向かって外側から開始し、関心と腺や小さな鋭いはさみと皮膚の間のカットの乳腺を持ち上げます。あなたが慎重にカットを確認して、皮膚や筋肉のない部分が腺に残らないように。すべての乳腺（図1）はこのプロトコルを使用して削除されますが、腹部乳腺（＃4）全体のマウント用に最適ですできます。 2番と3番目の腺は、いくつかの櫛筋肉を持っており、それらが全体のマウントおよび組織学のために使用することができますが、それらはすべての分析に適していない可能性があります。 </li></ol> 2。全体のマウント<ol><li>解剖前に少なくとも一日は、1リットルの三角フラスコ中で20分間蒸留水と沸騰の500mlのアルミニウム硫酸カリウムのカルミンおよび2.5Gの1gを溶解することによってカルミンのミョウバン溶液を調製。水で500 mlに最終的な音量を調整します。ワットマン濾紙を通して濾過し、防腐剤としてチモールの少量（数粒）を追加します。冷蔵。染色した後、カルミンのミョウバンのソリューションは、株式のボトルに戻って注ぎ、数週間にわたって何度も再利用することができます。色が薄くなると新鮮な溶液を作る。 </li><li>マウス（ステップ1）から乳腺を切除した後、スライドガラス上に直接それを広げる。 その場の形で元のを維持し、可能な限りそれを平らにしてみてください。適切にストレッチするときに腺がうまくスライド上に残ることでしょう。 </li><li> jar内の室温ではドラフト内で4時間、カルノア固定液（6時03分01秒100パーセントエタノール、クロロホルム、氷酢酸）で、スライドで、それを浸漬することによって腺を修正。他の人が十分であると思われる2時間を使用していることに注意してください。また、腺はまた、4℃で一晩固定することができます。 </li><li> 15分間70％エタノールで洗う。その後、瓶からエタノール溶液の半分を除去してのddH 2 Oで置き換えることによってそれを水に徐々に再水和する5分間インキュベートする。 3回繰り返します。各5分間、エタノールと水の風呂 - 、 - 35％ - 15％あるいは、70％を使用してください。 </li><li> 5分間蒸留水ですすいでください。 </li><li>室温で一晩カルミンのミョウバンで染色。それは染色は腺の厚さに応じて時間がかかる可能性があることに留意すべきである。 </li><li> 、再利用と徐々にキシレンに5分ごとにと明確なために、またはそのようなヒストグラムをクリアのような非毒性の代替のシリアルエタノール浴（50％、70％、95％、100％）によって染色した組織を脱水して染色カーミンのミョウバンを削除します。一晩。脱水とクリアどちらも厚い試料でより長いインキュベーションが必要になる場合があります。 </li><li>サリチル酸メチルの乳腺を浸し。画像が撮影できるようになるまで乳腺はドラフト内でサリチル酸メチルに保管することができます。 </li><li>画像はライトテーブルの上にスライドを配置することにより、（可能な場合は、&quot;マクロ&quot;機能を使用）三脚上に位置する通常のデジタルカメラでドラフト内で撮影することができます。高倍率のための解剖顕微鏡を使用してください。また、乳腺は、画像が十分に薄いマウントを提供する任意のイメージングプラットフォームで化学ドラフト外で撮影することを可能にする（ステップ2.7の後）などPermountなどのメディアに取り付けることができます。 </li><li>全体のマウントを使用して乳腺の発達を定量化するために、このようなダクトの部分に沿って分枝の数（またはサイド支店）などの測定、主ダクトの長さ、または脂肪組織の領域への上皮の比率を評価することができます。最後に、写真の説明書の後にtation、乳腺の全体のマウントは、切片と、従来の組織染色用パラフィンに埋め込むことができます。 </li></ol> 3。オキシトシン誘発の射乳<ol><li>それらが供給された直後にダムから子犬を区切ります。アッセイを実行する前に1時間を待つ。 </li><li>動物からそれらを削除せずに、母を犠牲にし、前に説明したように乳腺を公開する（ステップ1.1から1.8）。 </li><li>トランスファーピペットを使用して、均等に直接興味の乳腺にPBSまたはオキシトシンのソリューションのいずれかをドロップすることでアッセイを開始します。用量の大きいスペクトルを使用することができます。〜8 pg / mlの（生理的用量は）一般的に使用されます。しかし、高い非生理的な投与量は（最大1 mg / ml）を遺伝子組み換えマウスモデルにおける応答がないことを保証するために使用することができます。この手順では、それは射乳（オキシトシンの露出）をテストするコントロール（PBSにさらされる）と反対側のように片側を使用して、胸部（＃2-3）腺を使用することをお勧めします。 </li><li> 1分間インキュベートし、慎重に転送ピペットでそれを吸引することによってソリューションを削除する。ダクトへのミルクのエントリは、ビデオテープの録音によって、またはPBSまたはオキシトシン曝露前と後の画像を取ることによって監視できます。 </li></ol> 4。代表的な結果： 良い全体のマウントでは、乳腺がきちんと広がっていると上皮のダクトは簡単に（図2A）観測されています。腺が十分に広がっていない場合は固定液に入れたとき、それは部分的にまたは完全にスライドから持ち上げることができる。腺は、（図2B）、ハードと使用できなくなります。同様に、腺がよく解剖されていない場合、どちらかの上皮の一部が欠落して（図2C）や皮膚の部分を残りや腹部の筋肉は、腺の分析（図2D）と干渉することができます。 オキシトシンアッセイのために、それはあらゆる機会に、同じ時間のためのダムから哺乳仔を分離することが重要です。この方法では、あなたは牛乳やミルクの少量が前にオキシトシン曝露にダクトに存在しないことが確認されますが、また十分な母乳が筋上皮の収縮（図3A）によりダクトに放出することができる肺胞に存在すること。子犬があまりにも早く削除されると、牛乳は肺胞にし（図3B）を監視することは困難オキシトシンの効果をレンダリングするダクトに蓄積することができます。また、実験は子犬を除去した後早すぎるが実行されている場合、ミルクは肺胞に蓄積する時間がないし、筋上皮細胞のオキシトシン誘発性収縮は、（図3C）が観察されるダクトに十分なミルクのリリースをトリガしません。 <img src="/files/ftp_upload/2828/2828fig1.jpg" alt="図1"/> 図1マウスの乳腺の解剖。四肢（）でその背中の上でマウスをピン。腹から首への最初の皮膚を切り取ります。次に、点線（B）で示すように、各肢に腹の中心から垂直切開を行います。優しくピール動物の側の皮膚や乳腺（C）を公開し、それを固定する。 <img src="/files/ftp_upload/2828/2828fig2.jpg" alt="図2"/> 図2。マウス乳腺の全体のマウント。上皮管（矢印）とリンパ節（矢印）が容易に腺のうまく広がる乳腺の全体のマウント＃4（）で観察されています。悪い広がる乳腺が（B）が使用できなくなる、スライドから切り離さと崩壊になることがあります。不適切に摘出した乳腺のいずれか（D）、残りの足りない部分（C）または筋肉や皮膚の部分を持つことができます。 2番と3番目の腺が筋肉を櫛歯しているが、依然として全体のマウントまたは組織学的に使用することができることに注意してください。 <img src="/files/ftp_upload/2828/2828fig3.jpg" alt="図3"/> 図3。オキシトシン誘発の射乳がオキシトシンの曝露（）以下の（矢印）上皮のダクトで観察することができます。しかし、ダクトはすでに子犬、オキシトシン曝露後のダクトの牛乳のさらなる蓄積（矢印）の授乳最後の後の待ち時間の不適切に長い期間のためにアッセイの開始時に牛乳（矢印）で満たされている場合には難しい観察（B）。また、子犬授乳後の短い潜伏期間が適切にオキシトシン曝露（C）の後にダクト（矢印）で観察される肺胞に牛乳の十分な蓄積のために許可されません。画像は、数値のカメラ（サイバーショット、ソニー）を使用して、オキシトシン曝露の前後に採取した。 <img src="/files/ftp_upload/2828/2828fig4.jpg" alt="図4"/> 図4全体の取付けとオキシトシン治療アッセイの概略図。

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利害の衝突は宣言されません。

乳腺の発達と機能は密接にオーケストレーションされています。変異マウスは、腺のアーキテクチャ13,12を評価する必要性を強調する乳腺の発達と機能を損なうことができる遺伝子の変異を抱くことができる。乳腺の発達のすべての段階でこの資料に記載したように全体を取付けを行うことができます。典型的には、上皮の開発は思春期（〜6週齢）の途中でと2,10思春期（〜10...

<table border="1"><tbody><tr><th>試薬</th><th>会社</th><th>カタログ番号</th></tr><tr><td>カーマイン</td><td>シグマアルドリッチ</td><td> C1022 </td></tr><tr><td>アルミニウムカリウムの硫酸</td><td>シグマアルドリッチ</td><td> A6435 </td></tr><tr><td>チモール</td><td>シグマアルドリッチ</td><td> T0501 </td></tr><tr><td>メチルサリチル酸</td><td>シグマアルドリッチ</td><td> M6752 </td></tr><tr><td>オキシトシン</td><td>シグマアルドリッチ</td><td> O3251 </td></tr></tbody></table>

evaluation of mammary gland development and function in mouse models

人間の乳腺には、15〜20葉で構成されて乳首における分岐ダクトシステムの開口部に分泌するミルクその。これらのローブは分泌肺胞と収束ダクト1から作られたターミナルダクト小葉単位の数で構成そのものです。マウスでは、同様のアーキテクチャは、ダクトと肺胞が結合組織の間質内に散在されている妊娠で観察される。マウス乳腺上皮は細胞の2つの層、基底膜2の範囲で示される筋上皮細胞の外側の層に囲まれた管腔細胞の内側の層から構成されるダクトのシステムのようなツリーです。出生時に、唯一のrudimental膵管ツリーには、主ダクト15〜20支店で構成される、存在しています。ホルモン3,4,5の影響を受けて、約4週齢の思春期の開始時に枝の伸長と増幅の開始、、。 10週間で、間質のほとんどは、妊娠2日まで各性周期に分岐し、回帰のサイクルを受けることになるダクトの複雑なシステムに侵略されている。妊娠の開始時に、開発の第2段階は、肺胞6,7と呼ばれるブドウの形をしたミルクの分泌構造を形成する上皮の増殖と分化と、始まります。分娩に続いて、泌乳期間全体を通して、牛乳は内腔の分泌細胞で産生され、肺胞の内腔内に格納されています。仔の授乳によって誘発される神経反射によって刺激されたオキシトシン放出は、、牛乳はそれが子犬8〜使用可能になった乳首にダクトを介して輸送されるので、肺胞の周りやダクトに沿って筋上皮細胞の同期した収縮を誘発する。乳腺の発達、分化および機能はしっかりと画策し、必要とする、間質および上皮間だけでなく、上皮9,10,11内筋上皮および管腔細胞間の相互作用だけでなく、されています。これにより、これらの相互作用に関与する多くの遺伝子の変異は、妊娠初期、妊娠後期に分化し、授乳12,13につながる分泌活性化の間に思春期や肺胞形成時にどちら管伸長が損なわれる可能性があります。この記事では、我々はマウス乳腺を細かく分析し、全体のマウントを使用して開発を評価する方法について説明します。我々はまた、生体外オキシトシンベースの機能アッセイを用いて筋上皮の収縮と射乳を評価する方法を示します。乳腺の発達と機能に関する遺伝子変異の効果は、このように変異体と野生型コントロールマウスではこれら2つの手法を実行することにより、 その場で決定することができる。

Watch this Scientific Journal Video about Evaluation of Mammary Gland Development and Function in Mouse Models at JoVE.com

Evaluation of Mammary Gland Development and Function in Mouse Models

このメソッドは、マウスから乳腺の発達と機能を細かく分析し、評価する方法について説明します。射乳がオキシトシンベースの筋上皮細胞の収縮アッセイを用いて評価される間に切除された乳腺を全部マウントを使用して開発の度合いを評価されています。

マウスモデルにおける乳腺の発達と機能の評価

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

evaluation-of-mammary-gland-development-and-function-in-mouse-models

Research

JoVE Journal

Biology

36.9K ビュー.  University of Western Ontario. このメソッドは、マウスから乳腺の発達と機能を細かく分析し、評価する方法について説明します。射乳がオキシトシンベースの筋上皮細胞の収縮アッセイを用いて評価される間に切除された乳腺を全部マウントを使用して開発の度合いを評価されています。

ビデオ: Evaluation of Mammary Gland Development and Function in Mouse Models

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system is valuable for the pre-clinical in vivo evaluation of putative antitumor compounds.
The 4T1 cell line has been engineered to constitutively express the firefly luciferase gene (luc2). When mice carrying 4T1-luc2 tumors are injected with Luciferin the tumors emit a visual light signal that can be monitored using a sensitive optical imaging system like the IVIS Spectrum. The photon flux from the tumor is proportional to the number of light emitting cells and the signal can be measured to monitor tumor growth and development. IVIS is calibrated to enable absolute quantitation of the bioluminescent signal and longitudinal studies can be performed over many months and over several orders of signal magnitude without compromising the quantitative result.
Tumor growth can be monitored for several days by bioluminescence before the tumor size becomes palpable or measurable by traditional physical means. This rapid monitoring can provide insight into early events in tumor development or lead to shorter experimental procedures.
Tumor cell death and necrosis due to hypoxia or drug treatment is indicated early by a reduction in the bioluminescent signal. This cell death might not be accompanied by a reduction in tumor size as measured by physical means. The ability to see early events in tumor necrosis has significant impact on the selection and development of therapeutic agents.
Quantitative imaging of tumor growth using IVIS provides precise quantitation and accelerates the experimental process to generate results.

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Mammary tumor cells expressing luciferase are implanted subcutaneously in mice and visualized using optical imaging to monitor tumor growth and development non-invasively in a longitudinal study.

IVISスペクトラムを用いた乳腺腫瘍の in vivo生物発光イメージングに

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system ...

生物学

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

マウス乳腺上皮細胞は乳腺刺激分化の過程でMammospheresを形成し、

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

生体イメージングおよび卵巣癌の同所性マウスモデルにおける治療的処置の

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

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 ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

マウス唾液腺幹細胞の単離

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Mouse Sperm Cryopreservation and Recovery using the I&amp;middot;Cryo Kit

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

I ·クライオキットを使用してマウスの精子の凍結保存と回復

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

マウス乳腺に局在薬物送達のための管内注入

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

精製された野生型および変異型CFTRタンパク質の機能的再構成とチャネル活性の測定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

マウス乳腺の凍結切片の間接免疫

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

そのままに、全体のマウス乳腺の腺の形態と細胞外マトリックス式の分析のための分離

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...