著者は、動物との彼女の援助のために安定化siRNA溶液とヘザートービンを製造するための、マイケル·ゴールドバーグに感謝したいと思います。この研究は、DEIのDODイノベーター賞W81XWH-08-1から0659によって資金を供給された。 SKのポスドクトレーニングはスーザンGコーメン財団（KG101329）によってサポートされていました。著者は、回路図の作成のためのクリスティン·ジョンソンに感謝したいと思います。

1。注射液の調製<ol><li>リン酸緩衝生理食塩水（PBS）、または関心のある他の水溶液に0.2％エバンスブルー色素を準備します。この重要な染料は、管内注射の成功を評価するために有用であり、技術の専門知識を開発する際に視覚化助剤として推奨されます。 </li></ol>必要なボリュームを注入する腺の数および位置に依存するであろう。雌マウスは全て10乳腺を注入することができるが、小型化による腺の大きさに、グランドペア1および5は通常、溶液10μlを注射する。他のすべての乳腺のペアは20μLを注入している。これらのボリュームは、全体の乳管樹を満たすのに十分である。 2。術前の準備<ol><li>各マウスの体重を記録します。すべての前臨床試験と同様に、動物の体重は、潜在的な毒性を評価するために（週二回以上）を定期的に監視する必要があります。 </li><li>男を麻酔ウーズイソフルランチャンバーを用いて、目の潤滑剤を塗布。手順の間にマウスはノーズコーンを経由して酸素イソフルランの吸入2〜4％濃度を用いて麻酔され続けます。丁寧に応じてイソフルランのレベルを調整する、呼吸数の変化のためのマウスを監視します。 </li><li>皮下前鎮痛などの手続きにメロキシカム（5月10日MG / kg）を注入する。 </li><li>乳首エリアに店頭脱毛クリームを適用します。 5分待ってから、ゆっくりと円を描くように綿先端アプリケーターで抜け毛を取り除く。ぬるま湯で湿らせ、湿ったペーパータオルを使用してクリームを取り除きます。シェービングが原因乳首を損傷するリスクにはお勧めしません。 </li><li>そっと手足を下にテープでステレオスコープの下にマウスを固定します。 </li><li>アルコール綿棒で掃除注射部位。 </li></ol> 3。管内注射<ol><li>ステレオスコープの下に注入されるべき適切な乳首の位置を確認します。細かいマイクロ解剖気取っを使用乳首の開口部を覆う任意の古い角質を除去するzers。 </li><li>貼付33gの金属ハブ針で50μLシリンジにロードされた注射液の10〜20μL。時々、注射の後、少量（1μL以下）針を引き抜く際に乳首から漏出することができます。したがって、潜在的な損失を考慮して、それぞれ11または21μLを注入することをお勧めします。 </li><li>細かいピンセットでそっと乳首を持って、注射のためにそれを配置するために、わずかに持ち上げます。それは乳首を切断する必要はありません。 </li><li>急速乳管ルーメン内の流体を移動させることによって生じる潜在的な損傷を最小限に抑えるために徐々に溶液を注入する。注入速度は、約40μlの/分に維持されるべきである。 </li></ol> 4。術後処置<ol><li>注射部位を観察します。乳首領域または周囲の組織への外傷の兆候があってはならない。乳首の周辺の腫れは、おそらく乳房脂肪パッド注入早咲きのを示している成功管内注射よりR。 </li><li>ノーズコーンから動物を削除して、再利用するための分別のケージに移動します。低体温症を予防し、回復を支援するために加熱ランプ下のケージを置きます。マウスを単独で収容され、それらが意識と運動性を取り戻すまで、厳密に監視されます。 </li></ol> 5。乳腺組織の分析<ol><li>研究の完了時に、マウスを、隔離室で圧縮されたCO 2ガスを以下の頸椎脱臼により安楽死させる。乳腺切除され、ホールマウント調製物11、組織学、またはRNAおよびタンパク質の単離のために使用することができる。 </li></ol>

<ol>
	<li>Dooley, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ductal+lavage+for+detection+of+cellular+atypia+in+women+at+high+risk+for+breast+cancer.">Ductal lavage for detection of cellular atypia in women at high risk for breast cancer.</a> J Natl Cancer Inst. 93, 1624-1632 (2001).</li><li>Jacobs, L., Sukumar, S., Stearns, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraductal+therapy+for+the+prevention+of+breast+cancer.">Intraductal therapy for the prevention of breast cancer.</a> Curr Opin Investig Drugs. 11, 646-652 (2011).</li><li>King, B. L., Love, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intraductal+approach+to+the+breast:+raison+d'etre.">The intraductal approach to the breast: raison d'etre.</a> Breast Cancer Res. 8, 206 (2006).</li><li>Murata, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ductal+access+for+prevention+and+therapy+of+mammary+tumors.">Ductal access for prevention and therapy of mammary tumors.</a> Cancer Res. 66, 638-645 (2006).</li><li>Stearns, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+and+clinical+evaluation+of+intraductally+administered+agents+in+early+breast+cancer.">Preclinical and clinical evaluation of intraductally administered agents in early breast cancer.</a> Sci Transl Med. 3, 106ra108 (2011).</li><li>Flanagan, M., Love, S., Hwang, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Status+of+Intraductal+Therapy+for+Ductal+Carcinoma+in+Situ.">Status of Intraductal Therapy for Ductal Carcinoma in Situ.</a> Curr Breast Cancer Rep. 2, 75-82 (2010).</li><li>Nguyen, D. -. A., Beeman, N., Lewis, M., Schaack, J., Neville, M. C., Ip, M. M., Asch, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in mammary gland biology and breast cancer research. , 259-270 (2000).</li><li>Behbod, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+intraductal+human-in-mouse+transplantation+model+mimics+the+subtypes+of+ductal+carcinoma+in+situ.">An intraductal human-in-mouse transplantation model mimics the subtypes of ductal carcinoma in situ.</a> Breast Cancer Res. 11, R66 (2009).</li><li>Bu, W., Xin, L., Toneff, M., Li, L., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus+vectors+for+stably+introducing+genes+into+mammary+epithelial+cells+in+vivo.">Lentivirus vectors for stably introducing genes into mammary epithelial cells in vivo.</a> J Mammary Gland Biol Neoplasia. 14, 401-404 (2009).</li><li>Reddy, J. P., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RCAS-TVA+system+for+introduction+of+oncogenes+into+selected+somatic+mammary+epithelial+cells+in+vivo.">The RCAS-TVA system for introduction of oncogenes into selected somatic mammary epithelial cells in vivo.</a> J Mammary Gland Biol Neoplasia. 14, 405-409 (2009).</li><li>Plante, I., Stewart, M. K., Laird, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+mammary+gland+development+and+function+in+mouse+models.">Evaluation of mammary gland development and function in mouse models.</a> J. Vis. Exp. , e2828 (2011).</li></ol>

著者は、財政的な開示がない。

トランスジェニックおよびノックアウトマウスは、乳癌における個々の遺伝子のインビボでの役割を研究するための非常に貴重なツールである。しかしながら、これらの動物を生成するために、コストと時間がかかり、遺伝子ノックダウンは、時には乳腺上のその遺伝子の特異的な効果を認識するから私たちを排除する通常ユビキタスである。したがって、標的ノックダウンは、他の器官における非特異的な副作用および毒性の問題を軽減するであろう。 ここに提示されたsiRNAの管内注入は、マウスの乳腺の局所的な遺伝子ノックダウンを可能にします。これは、siRNA送達に限定されるものではなく、乳腺を標的とし、局所的な薬物送達のための迅速な方法である。このような乳腺特異的薬物送達は、他の器官への非特異的な副作用および毒性を回避し、乳腺発達および乳房腫瘍形成の間の特定の時点での遺伝子変化の研究を可能にする。済州の証拠はない非注射腺中のRNAの粒子、肝臓やこのメソッドは乳腺の局所的な遺伝子ノックダウンのための好ましい方法を提示していることのさらなる証拠を提供する他の臓器。さらに、管内注射は、治療剤の長期モニタリングを可能にするために数ヶ月にわたって毎週または隔週に繰り返すことができる。 この技術は、マウス乳腺管に様々な試薬の管内送達をアッセイするための可能性を開く。よく特徴付けられたマウスモデルにおける局所的送達における進歩は、非侵襲性の適用を加速する必要があり、ヒトにおける治療戦略の標的化。

マウス乳腺腺は乳首の中央ダクトに収束する複雑な乳管肺胞ツリーで構成されている。マウスだけでなくヒトでは、乳房の腫瘍の大部分は、乳管の内側を覆う上皮細胞に由来する。現在利用可能な治療は、静脈内化学療法、放射線、手術が含まれる。ローカライズされた管内の治療は、1〜3を探検し、減少した毒性や副作用の点で重要な利点を提供しています。これらは、より広く適用することができる前に、しかし、いくつかの課題が残っている。マウス乳腺腺で同等の手順を実行する能力は、管内介入の特徴付けを容易にする。ここでは、直接マウス乳腺ダクト内に試薬を送達するための非侵襲的方法を提示する。 乳癌患者およびマウスモデルの両方における化学療法剤の管内送達は、以前の証拠を有する高度に有効であることが示された全身毒性や長期の組織病 ​​理学的には3-6に変更されます。さらに、腫瘍細胞注射と癌遺伝子のレンチウイルスベクター送達は、管内噴射7-10を介して実現可能であることが以前に示されている。 薬物送達に加えて、この手順は、siRNAの管内注入による乳腺遺伝子のサイレンシングローカルに有用である。 siRNA溶液の小容量の注入は乳腺の解剖学と生理学を変更することなく、効果的である。当研究室では最近、自然発生的に（Brockさんら 、未発表）腫瘍進行の予防になり乳腺腫瘍を発症するトランスジェニックマウスでの重要なメディエーター遺伝子の正常な抑制を示している。私たちは、目に見える組織損傷または毒性を2週間間隔で7回まで同じ乳首に隔週注射を繰り返すことができました。 管内注射は開発に取り組むための成体マウスでタイムアウトすることができます妊娠、授乳、そして退行中または腫瘍の発生および進行の間に、後の段階をターゲットに乳腺発達に関連するopmentally固有のイベント。 8週齢の幼い処女雌は、私たちの研究室で非侵襲的に注入されている。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>isoflurane</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>2-4%</td>
		</tr>
		<tr>
			<td>meloxicam</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>5-10 mg/ml</td>
		</tr>
		<tr>
			<td>Evans blue dye</td>
			<td>Sigma-Aldrich</td>
			<td>E2129-10G</td>
			<td>0.2% in PBS</td>
		</tr>
		<tr>
			<td>hair removing cream</td>
			<td>Veet</td>
			<td>&nbsp;</td>
			<td>Choose sensitive option</td>
		</tr>
		<tr>
			<td>stereoscope</td>
			<td>Zeiss</td>
			<td>Stemi DV4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>micro-dissecting tweezers</td>
			<td>Roboz</td>
			<td>rs-4976</td>
			<td>With a 0.10 x 0.06 mm tip made of dumostar</td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Hamilton</td>
			<td>7637-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>33-gauge needle</td>
			<td>Hamilton</td>
			<td>7803-05</td>
			<td>point style 4, 12 degree bevel (standard)</td>
		</tr>
		<tr>
			<td>siGLO cyclophilin B control siRNA</td>
			<td>Dharmacon</td>
			<td>D-001610-01-05</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

intraductal injection for localized drug delivery to the mouse mammary gland

ここに私たちは、管内注射を経由して、マウスの乳腺への様々な試薬を提供するためのプロトコルを記述します。局所的な薬物送達およびノックダウン乳腺上皮内の遺伝子のは、そのような妊娠や授乳などの発達段階とは無関係である、適切な標的化分子の不足のために達成することは困難であった。ここで、我々はマウスの乳首に管内注射によって成人期にどの段階で乳腺に試薬の局所送達するための技術について説明します。注射は、麻酔下で、生きたマウスで行い、乳腺に対して非侵襲的で局所的な薬物送達を可能にすることができる。さらに、注射は、ニップルを損傷することなく、数ヶ月にわたって繰り返すことができる。このようなエバンスブルーなどの重要な染料は、テクニックを学ぶことは非常に有用である。青色色素の管内注入すると、全体の乳管ツリーは、目に見えるようになる。さらに、蛍光標識された試薬も可能にする乳腺内の可視化と配布。この技術は、siRNA、化学療法剤、および小分子を含む種々の化合物に適応可能である。

乳首を簡単に髪が（ 図1A）、周辺エリアでは削除された後、実体顕微鏡を使用してローカライズできます。注入技術を習得するためには、エバンスブルー色素を注入し、腺（ 図1BおよびC）の完全性を監視することをお勧めします。このアプローチは、一方が視覚的に色素が全体管系（ 図1Cおよび図2A）に達したか否かを評価することができるように、各腺に注入されるべき適切な量の決定を可能にする。注入されたエバンスブルー画像の画像は、全体乳管ツリーがカーミンミョウバン色素（ 図2B）で染色された全装着乳腺、比較することができる。 この注入法の堅牢性がオペレータに大きく依存していることに注意してください。例えば、ダクトを穿孔すると、乳腺脂肪注入になりますし、溶液を注入することは速すぎて管上皮CELが損傷する恐れがありLSおよび炎症反応を引き起こす。成功した管内注入は乳腺に局所的な薬物送達を可能にし、多くの場合そうでなければ観察される非特異的な副作用を減少させる。 蛍光を注射した腺の画像例を図3に示すシクロフィリン（非必須遺伝子）を標的とするsiRNAタグ。 <img alt="図1" src="/files/ftp_upload/50692/50692fig1.jpg" /> 図1。エバンスブルー色素と、ニップルを介して注入鼠径乳腺。 20μlのエバンスブルー色素注射後の注射とBの前に乳首のA）外観）。腫脹または組織の損傷は、皮膚を開くとします。C）観察されない、染料が全体の乳管ツリーの可視化を可能にする。 <img alt="図2" src="/files/ftp_upload/50692/50692fig2.jpg" /> 図2。エバンスブルーインの分析をホールマウントjected腺。 A）動物から切除し、すぐに脂肪後カルミン色素と同じ腺の注入。B）のホールマウント染色した後、スライドガラス上に広げ解剖し腺の代表画像がクリアされていた。 A）との比較では、注入されたソリューションは、乳管ツリーを満たし、腺は解剖学的に無傷であることを確認する。 <img alt="図3" src="/files/ftp_upload/50692/50692fig3.jpg" /> 図3。蛍光siRNAの管内配信。新鮮な組織サンプルの代表的なイメージは、48時間注入後に切除した。

Watch this Scientific Journal Video about Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland at JoVE.com

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

マウス乳腺水性試薬の非侵襲管内送達のためのプロトコルが記載されている。この方法は、特に乳管を対象とした乳腺の乳首にローカライズ注入を利用しています。この技術は、siRNA、化学療法剤および小分子を含む化合物の様々な適合可能である。

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 ...

intraductal-injection-for-localized-drug-delivery-to-mouse-mammary

Research

JoVE Journal

Biology

34.6K ビュー.  Boston Children's Hospital and Harvard Medical School. マウス乳腺水性試薬の非侵襲管内送達のためのプロトコルが記載されている。この方法は、特に乳管を対象とした乳腺の乳首にローカライズ注入を利用しています。この技術は、siRNA、化学療法剤および小分子を含む化合物の様々な適合可能である。 

ビデオ: Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

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 ...

生物学

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 ...

Evaluation of Mammary Gland Development and Function in Mouse Models

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 composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

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 ...

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once the pre-cursor round cell begins to elongate and differentiate into what is morphologically recognized as a spermatozoon. However, the spermatozoon is very immature, having no ability for motility or egg recognition. Both of these events occur once the spermatozoa transit a secondary organ known as the epididymis. During the ~12 day passage that it takes for a sperm cell to pass through the epididymis, post-translational modifications of existing proteins play a pivotal role in the maturation of the cell. One major facet of such is protein phosphorylation. In order to characterize phosphorylation events taking place during sperm maturation, both pure sperm cell populations and pre-fractionation of phosphopeptides must be established. Using back flushing techniques, a method for the isolation of pure spermatozoa of high quality and yield from the distal or caudal epididymides is outlined. The steps for solubilization, digestion, and pre-fractionation of sperm phosphopeptides through TiO2 affinity chromatography are explained. Once isolated, phosphopeptides can be injected into MS to identify both protein phosphorylation events on specific amino acid residues and quantify the levels of phosphorylation taking place during the sperm maturation processes.

Proteomic analysis of any cell type is highly dependent on both purity and pre-fractionation of the starting material in order to de-complexify the sample prior to liquid chromatography mass spectrometry (MS). By using back-flushing techniques, pure spermatozoa can be obtained from rodents. Following digestion, phosphopeptides can be enriched using TiO2.

齧歯類精巣上体精子のリン酸化ペプチドの分析

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once ...

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 ...

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. The first procedure consists of perforation of the tympanic bulla, termed bullostomy; the second one is a transtympanic injection. Both emulate human clinical intratympanic procedures.
Chitosan-glycerophosphate (CGP) and Ringer&#180;s Lactate buffer (RL) were used as biocompatible vehicles for local drug delivery. CGP is a nontoxic biodegradable polymer widely used in pharmaceutical applications. It is a viscous liquid at RT&#160;but it congeals to a semi solid phase at body temperature. RL is an isotonic solution used for intravenous administrations in humans. A small volume of this vehicle is precisely placed on the Round Window (RW) niche by means of a bullostomy. A transtympanic injection fills the middle ear and allows less control but broader access to the inner ear.
The safety profiles of both techniques were studied and compared by using functional and morphological tests. Hearing was evaluated by registering the Auditory Brainstem Response (ABR) before and several times after microsurgery. The cytoarchitecture and preservation level of cochlear structures were studied by conventional histological techniques in paraformaldehyde-fixed and decalcified cochlear samples. In parallel, unfixed cochlear samples were taken and immediately frozen to analyze gene expression profiles of inflammatory markers by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR).
Both procedures are suitable as drug delivery methods into the mouse middle ear, although transtympanic injection proved to be less invasive compared to bullostomy.

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

Two microsurgery approaches for local drug delivery to the inner ear are described here and compared in terms of impact on hearing parameters, cochlear cytoarchitecture and expression of inflammatory markers.

マウス内耳に標的薬の配布方法の比較研究：Bullostomy<em&gt;対</em&gt; Transtympanicインジェクション

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. ...

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 ...

Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile duct proliferation. However, long-term experiments can lead to increased morbidity and mortality. In select mouse strains with underlying liver disease, meaningful comparisons can be made even with ligation of a single lobe of the liver, which can reduce animal losses and expenses. Here, we describe partial bile duct ligation (pBDL) in the mouse, in which only the left hepatic bile duct is ligated, causing biliary obstruction in the left lobe but not the remaining lobes. With careful microsurgical technique, pBDL experiments can be cost-effective, since the unligated lobe serves as an internal control to the ligated lobes, when subjected to the same conditions in the same animal. Unlike cBDL, a separate sham-operated control group is not necessary. pBDL is highly useful to directly compare localized versus systemic effects of cholestasis and other retained bile components. pBDL can also be repurposed as a novel method to investigate mechanisms related to medications and cell migration.

Here, we present partial bile duct ligation as a surgical model of liver injury and regeneration in rodents.

マウスの部分胆管結紮: ローカライズされた閉塞性胆汁うっ滞の制御モデル

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

トランス内部細胞のキメリズム率が高いため、胚性幹細胞の大量注入

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Bovine Mammary Gland Biopsy Techniques

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, signaling pathways, and protein translation. This article describes two techniques for biopsy of the bovine mammary gland (MG). Three healthy Holstein dairy cows were the subjects. Before biopsies, cows were milked and subsequently restrained in a cattle chute. An analgesic (flunixin meglumine, 1.1 to 2.2 mg/kg of body weight) was administered via jugular intravenous [IV] injection 15-20 min prior to biopsy. For standing sedation, xylazine hydrochloride (0.01-0.05 mg/kg of body weight) was injected via the coccygeal vessels 5-10 min before the procedure. Once adequately sedated, the biopsy site was aseptically prepared and locally anaesthetized with 6 mL of 2% lidocaine hydrochloride via subcutaneous injection. Using aseptic technique, a 2 to 3 cm vertical incision was made using a number 10 scalpel. Core and needle biopsy tools were used. The core biopsy tool was attached to a cordless drill and inserted into the MG tissue through the incision using a clock-wise drill action. The needle biopsy tool was manually inserted into the incision site. Immediately after the procedure, an assistant applied pressure on the incision site for 20 to 25 min using a sterile towel to achieve hemostasis. Stainless steel surgical staples were used to oppose the skin incision. The staples were removed 10 days post-procedure. The main advantages of core and needle biopsies is that both approaches are minimally invasive procedures that can be safely performed in healthy cows. Milk yield following the biopsy was unaffected. These procedures require a short recovery time and result in fewer risks of complications. Specific limitations may include bleeding after the biopsy and infection on the biopsy site. Applications of these techniques include tissue collection for clinical diagnosis and research purposes, such as primary cell culture.

This article presents a bovine mammary gland biopsy using core and needle biopsy tools. Harvested tissue can be used for cell culture or to assess mammary physiology and metabolism including gene expression, protein expression, protein modifications, immunohistochemistry, and metabolite concentrations.