1。移植上皮の準備新鮮または凍結ドナー上皮組織はクリア脂肪パッドに移植したが、凍結ドナー上皮の事前準備は、移植効率の最小限の低下（&gt; 85％の効率）とより便利ですできます。次のように凍結ドナー上皮の準備のためのプロトコルは、次のとおりです。 ドナーの移植の上皮を準備する前に、cryovialsに1ミリリットル約上皮、アリコートのために凍結するメディアを準備する。必要になるまでこれは、6ヶ月までは4℃で保存することができます。凍結メディアはDMEM/F12 HEPESおよびNaHCO 3で緩衝培地、10％ウシ胎児血清（FBS）および10％ジメチルスルホキシド（DMSO）が含まれています。 最近安楽死させた雌マウスから乳腺上皮を採取。 <ol><li> 70％EtOHでマウスを洗ってください。 </li><li>腹側体腔の漿液膜を貫通することなく、恥骨から胸骨に至る体壁を通して半ば矢状切断してください。各後肢（図1）に向かって恥骨で最初の切開から斜めのカットを行います。 </li><li>体壁の裏側に筋膜に接続された第 5 回鼠径部乳腺を公開するために離れて漿液膜から体壁ピール。これを達成する最良の方法はないピアス腹側の体腔に注意して、曲がったピンセットの湾曲部を慎重に漿液膜を把握することです。同時に、直線鋸歯状の鉗子と外皮のカットエッジをつかみ、漿液膜から体壁を引き出します。 </li><li>乳腺内のリンパ節を特定する。リンパ節は、乳腺の3つの主要な血管の交差点に位置し、脂肪パッド（図2）内に密度の高い小結節として感じることができる。目のはさみの先端で、ニップノードを取り巻く脂肪組織。皮膚の表皮側の人差し指で、脂肪パッドのノードの出力を高め、湾曲したピンセットでそれを挟まないように圧力を適用する。リンパ節を捨てる。 </li><li>皮膚のマージンを押しながら、曲がったピンセットの湾曲した縁で、徐々に体壁から脂肪パッドピール乳腺脂肪体を把握する。あなたが一体で全体乳腺を除去するように、マウスの背側表面に向かって脂肪パッドの方向に従ってください。 </li><li>滅菌プラスチックの切削ブロックで乳腺を置く。それは湿った保つために腺の表面に、200μlの程度、小容量のリムーバブルメディアを追加。新鮮なカミソリの刃で1mmの部分に腺を飾らない。 4つの部分にみじん切りの腺を分割し、鉗子で凍結メディアの4つのcryovials間で配布する。外側のコンパートメントにイソプロピルアルコールの250ミリリットルいるNalgene&quot;ミスターフロスティ細胞の冷凍庫にcryovialsを置く。24時間70℃以下の冷凍庫でNalgeneの冷凍庫に入れて。ために必要になるまで液体窒素中に乳腺の断片の凍結バイアルを保存移植各クライオバイアルは4〜8宿主マウスのクリア脂肪パッドに移植するのに十分な乳腺のフラグメントが含まれます。 </li></ol> 2。移植細胞の調製私たちの研究室では、脂肪パッドをクリアマウスに不死化乳腺上皮細胞株、乳腺腫瘍細胞株またはプライマリ乳腺細胞を含むさまざまな種類の細胞を、移植しています。次のように移植するためのカンマ- D細胞の調製のためのプロトコルは、次のとおりです。 <ol><li>コンマ- D細胞は、DMEMを含む正規MECL培地で培養されています：F12、2％アダルト牛血清（ABS）、mEGF、インスリン、抗生物質/抗真菌剤（AB / AM）を補充した。 </li><li>収穫のカンマ- D細胞が85％-90％コンフルエントの細胞。 </li><li>細胞をペレットに5分間1200rpmで細胞懸濁液を遠心してください。新鮮なMECLメディアで細胞を再懸濁し、5 × 10 6個 / mlに細胞濃度を調整する。 </li></ol>主な乳腺細胞の移植のために、主要な乳腺上皮細胞はマウス5 番目の乳腺からの機械的および酵素的解離によって分離されています。 F12のメディアをして所望の濃度に調整し、通常5 × 10 6個 / ml：細胞は、DMEMに再懸濁する。 3。マウス乳腺脂肪体をクリアするための標準手順<ol><li>三週間、古いホストのマウスを麻酔。 &lt;12 gが乳管の成長が開始されていないことを保証するためであるマウスを選択します。我々は、2,2,2 - トリブロムエタノールの体重から200mg/kg使用。 </li><li>マウスに麻酔をした後に腹部が愚弄になるように、しっかりと手術のステージで、広げた手足と、腹側にマウスを抑制する。手術前に、鎮痛薬を管理し、ブピバカイン（&lt;1mg/kg）またはブプレノルフィン（0.05mg/kg）のどちらか、皮下。 </li><li>数ミリの骨盤上記鉗子で皮膚をつかみ、腹部からそれを持ち上げる<html> 。ニップは、はさみで皮膚と恥骨から胸骨に至る体壁を通して半ば矢状カットを作るために。腹側体腔のピアス漿液膜はしないでください。創傷クリップが排尿するためにマウスの能​​力と干渉しないように、あまりにも尿道の近くに最初の切開を行わないでください。 </li><li>最初の切開から、各後肢に向かって骨盤全体の半ばラインから皮膚を通して斜めのカットを行います。 </li><li>優しく漿液膜を把握するために湾曲鉗子の湾曲した領域を使用します。皮膚のカットマージンをつかみ、漿液膜から体壁を引くと体壁の下側の筋膜上にある5 番目の鼠径部乳腺を公開する組織鉗子を使用してください。 </li><li>ニップル、ノードとノード（図2）に先頭に2つの動脈を焼灼する</li><li>第四および第五腺を区切ります。 6 番目の腺から内部成長を防ぐために、第五腺のマージンを焼灼する。 </li><li>ノードの（ニップルに向かって側）だけで腹をはさみで脂肪パッドを横断する4つ目の乳腺の脂肪パッドを割ります。しっかりと脂肪体の腹側部分を把握し、一枚で体壁からそれを引き離しに曲がったピンセットを使用してください。つのスライド間で脂肪体の腹側部分を押すと、カルノア固定液を含むガラスの組織学的染色皿に入れ。この組織は、クリアマージン（図3）を確認するために固定されており、カルミンのミョウバン液で後染色されます。湾曲鉗子で乳腺のすべての痕跡を除去する脂肪パッドの除去部分の領域の周囲の筋膜を清掃してください。 </li></ol> 4。脂肪パッドをクリアするための代替&quot;スペアリングの手順いずれかのマウス乳腺脂肪パッドをクリアするための標準手順で有能になった後、彼らはブリルら （2008）2によって開発された低侵襲温存の手順を試みることを好むことがあります。 <ol><li>標準的な手順と同様にマウスを準備します。 </li><li>第四鼠径乳腺の乳首をつかみ、ハサミの先端で乳首の周りに3〜5ミリメ​​ートルの円周の円をカット。 </li><li>皮膚のカット部分を切り離し、穴から第四鼠径部乳腺を引き出すために離れて身体から乳首を引っ張る。 </li><li>ノードを識別します。スルーホールノードと6 番目の乳腺を焼灼するために焼灼を使用しています。 </li><li>単にノードの前に第 5 回乳腺の最も表面的な部分をカットして切り離す。ニップル、上皮を含む乳腺脂肪体の皮膚と部分の円形の領域が一体的に削除されます。 </li></ol> 5。組織/細胞移植<ol><li>上皮の破片、霜上皮のクライオバイアルを移植するとDMSOを希釈するために新鮮な培地を含むペトリ皿に上皮フラグメントを転送する。新しい上皮用はさみ先端とクリア脂肪パッドに小さな穴やポケットを作る。針の鉗子とポケットにドナーの上皮の1mmの部分を置く。組織の場所に残っているように、針の鉗子を撤回として組織鉗子で閉じてポケットホールド。 </li><li>細胞を移植するために、22秒計の針と50μlのハミルトンシリンジを用いて各脂肪パッドへのメディアの10μlに50,000個の細胞を注入する。 </li></ol> 6。閉会/マウスのリバイバル<ol><li>閉会のスキンを手配。皮膚のマージンを反対つかみ、、鉗子でそれらを一緒に押したまま腹体腔の漿液膜から離して持ち上げ、創傷クリップで閉じてください。一般的に、それは半ば矢状切開と標準的な手順については、各斜切開用のクリップを閉じるには、2つのクリップを使用する必要があります。尿道を閉塞することは避けてください。それは、縫合糸の半ばに矢状と斜切開の交差点で皮膚のギャップを埋めるために必要な場合があります。スペア手順は、各サイド用の創傷クリップが必要になります。 </li><li> 。彼女のケージにマウスを置いて、マウスが復活するまで加熱ランプの下にケージを保つ。 </li><li>創傷クリップは、2週間後に削除する必要があります。 </li></ol><img src="/files/ftp_upload/1849/1849fig1.jpg" alt="図1" /> 図1。切開パターンのデモンストレーション。 <img src="/files/ftp_upload/1849/1849fig2.jpg" alt="図2" /> 図2リンパ節、血管、乳頭および内因性上皮のロケーション。指定された場所で焼灼する。 <img src="/files/ftp_upload/1849/1849fig3.jpg" alt="図3" /> 図3。ホールマウントは、その内因性上皮が円内の領域に限定し、したがって、マージンは&quot;&quot;明確にされている証拠です。 7。代表的な結果_r.jpg&quot;ALT =&quot;図4&quot;/&gt; 図4。消去のマージンを示す3週齢のマウスから取り外した脂肪パッドのホールマウント。 <img src="/files/ftp_upload/1849/1849fig2_r.jpg" alt="図5" /> 図5。内因性の乳腺上皮のクリア脂肪パッドを示す10週齢のマウスからの全マウント。 <img src="/files/ftp_upload/1849/1849fig3_r.jpg" alt="図6" /> 図6 10週齢のマウスからクリア脂肪パッドのヘマトキシリンおよびエオシン染色切片。 <img src="/files/ftp_upload/1849/1849fig4_r.jpg" alt="図7" /> 図7。移植上皮から開発された正常な乳管ツリーを示す10週齢のマウスからの全マウント。 <img src="/files/ftp_upload/1849/1849fig5_r.jpg" alt="図8" /> 図8。移植上皮から開発された乳管のヘマトキシリンおよびエオシン染色切片。 <img src="/files/ftp_upload/1849/1849fig6_r.jpg" alt="図9" /> 図9。移植上皮から開発された乳腺腫瘍のヘマトキシリンおよびエオシン染色切片。

<ol>
	<li>DeOme, K. B., Faulkin, L. J., Bern, H. A., Blair, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+mammary+tumors+from+hyperplastic+alveolar+nodules+transplanted+into+gland-free+mammary+fat+pads+of+female+C3H+mice.">Development of mammary tumors from hyperplastic alveolar nodules transplanted into gland-free mammary fat pads of female C3H mice.</a> Cancer Res. 19, 515-520 (1959).</li><li>Brill, B., Boecher, N., Groner, B., Shemanko, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sparing+procedure+to+clear+the+mouse+mammary+fat+pad+of+epithelial+components+for+transplantation+analysis.">A sparing procedure to clear the mouse mammary fat pad of epithelial components for transplantation analysis.</a> Laboratory Animals. 42, 104-110 (2008).</li><li>Jerry, D. J., Kittrell, F. S., Kuperwasser, C., Laucirica, R., Dickinson, E. S., Bonilla, P. J., Butel, J. S., Medina, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mammary-specific+model+demonstrates+the+role+of+the+p53+tumor+suppressor+gene+in+tumor+development.">A mammary-specific model demonstrates the role of the p53 tumor suppressor gene in tumor development.</a> Oncogene. 19, 1052-1058 (2000).</li><li>Shackleton, M., Vaillant, F., Simpson, K. J., Stingl, J., Smyth, G. K., Asselin-Labat, M. -. L., Wu, L., Lindman, G. J., Visvader, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+functional+mammary+gland+from+a+single+stem+cell.">Generation of a functional mammary gland from a single stem cell.</a> Nature. 439, 84-88 (2006).</li><li>Stingl, J., Eirew, P., Ricketson, I., Shackleton, M., Vaillant, F., Choi, D., Li, H. I., Eaves, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+unique+properties+of+mammary+epithelial+stem+cells.">Purification and unique properties of mammary epithelial stem cells.</a> Nature. 439, 993-997 (2006).</li><li>Medina, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preneoplastic+Lesions+in+Murine+Mammary+Cancer.">Preneoplastic Lesions in Murine Mammary Cancer.</a> Cancer Res. 36, 2589-2595 (1976).</li><li>Wagner, K. -. U., Boulanger, C. A., Henry, S. g. a. g. i. a. s., Hennighausen, M., Smith, G. H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+adjunct+mammary+epithelial+cell+population+in+parous+females:+its+role+in+functional+adaptation+and+tissue+renewal.">An adjunct mammary epithelial cell population in parous females: its role in functional adaptation and tissue renewal.</a> Development. 129, 1377-1386 (2002).</li><li>Dunphy, K. A., Blackburn, A. C., Haoheng, Y., O Connell, L. R., Jerry, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+and+progesterone+induce+persistent+increases+in+p53-dependent+apoptosis+and+suppress+mammary+tumors+in+BALB/c-Trp53+/-+mice.">Estrogen and progesterone induce persistent increases in p53-dependent apoptosis and suppress mammary tumors in BALB/c-Trp53+/- mice.</a> Breast Cancer Res. 10 (3), R43-R43 (2008).</li></ol>

このレポートでは、我々は、乳腺上皮移植の準備のためにマウス乳腺脂肪パッドをクリアするための2つの代替方法を提示する。標準的な手順はDeOme らによる1959年に開発から使用されています。1は、より侵襲が、乳腺の構造と配向が容易に閲覧されているため、標準的な方法は、初心者を訓練するための好ましい方法です。しかし、温存手続きがあるため、いくつかの利点...

mammary epithelial transplant procedure

この記事ではDeOmeキロバイトら 1ブリルB らが開発温存手順によって開発された脂肪パッドのクリアランスの手順を説明し、比較する2、乳腺上皮移植の手続きが続く。それは乳腺上皮の著しい発展は、思春期後まで発生しないという事実が利用されるため乳腺移植の手順は、広く乳腺生物学者によって使用されます。生後3週で、乳腺上皮木の成長は、乳首の近傍に限定されていると脂肪パッドは、乳腺上皮の大部分を欠いているが、7週齢で上皮管ツリー全体脂肪体全体に拡張。したがって、上皮を含む脂肪パッドのこの小さな部分は、乳首やリンパ節の間の領域は、生後3週間で削除された場合、内因性上皮は乳腺脂肪体に移入することはありませんし、脂肪パッドは、&quot;クリアとして記述されています&quot;。この時点で、別のソースからの乳腺上皮は、それは脂肪体全体を通して乳管の木を拡張する可能性を秘めているクリア脂肪体に移植することができる。この手順は、決定、4,5限られた希釈で細胞を移植することにより、乳腺幹細胞の同定には、全体の動物の3遺伝子型の交絡影響することなくトランスジェニック乳腺上皮組織における腫瘍表現型の検査を含む多くの実験モデルで利用されている過形成結節は、乳腺腫瘍6に進みますし、乳腺上皮7,8の挙動に関する事前のホルモン暴露の影響を評価する場合。 三週齢の宿主マウスは、麻酔きれいにして外科的ステージに抑制。半ば矢状切開は恥骨から胸骨まで延びる、皮膚ではなく、腹膜を介して行われます。斜めカットは、それぞれの脚に向かって骨盤全体の半ばに矢状切開から皮膚を介して行われます。皮膚は4鼠径乳腺を公開するために離れて腹膜からプルされます。脂肪パッドは、リンパ節への脂肪パッドの組織の前方を削除することによってクリアされます。上皮のフラグメントまたは上皮細胞は、残りのクリア脂肪体に移植され、マウスが閉じられます。

Watch this Scientific Journal Video about Mammary Epithelial Transplant Procedure at JoVE.com

Mammary Epithelial Transplant Procedure

この記事ではDeOme KBで開発した手順を示しています<em&gt;ら。</em&gt;（1959）とブリルBによって開発されたスペア手順<em&gt;ら。</em&gt;（2008）乳腺の断片、乳腺上皮細胞、または乳腺腫瘍細胞の移植の準備のために思春期のマウスの4番目の鼠径部乳腺脂肪体をクリアする。

乳腺上皮移植の手順

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

Research

JoVE Journal

Biology

30.9K ビュー.  University of Massachussetts. この記事ではDeOme KBで開発した手順を示していますら。（1959）とブリルBによって開発されたスペア手順ら。（2008）乳腺の断片、乳腺上皮細胞、または乳腺腫瘍細胞の移植の準備のために思春期のマウスの4番目の鼠径部乳腺脂肪体をクリアする。

ビデオ: Mammary Epithelial Transplant Procedure

Investigating the Immunological Mechanisms Underlying Organ Transplant Rejection

免疫学的メカニズム深部臓器移植の拒絶反応の調査

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 ...

Dendra2 Photoswitching through the Mammary Imaging Window

In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution1-4 has resulted in important insights into mechanisms of metastatic behavior such as migration, invasion and intravasation of tumor cells5, 6, angiogenesis3 and immune cells response7-9. We have recently reported a technique to image orthotopic mammary carcinomas over multiple intravital imaging sessions in living mice10. For this, we have developed a Mammary Imaging Window (MIW) and optimized imaging parameters for Dendra211 photoswitching and imaging in vivo. Here, we describe the protocol for the manufacturing of MIW, insertion of the MIW on top of a tumor and imaging of the Dendra2- labeled tumor cells using a custom built imaging box. This protocol can be used to image the metastatic behavior of tumor cells in distinct microenvironments in tumors and allows for long term imaging of blood vessels, tumor cells and host cells.

Intravital photoswitching and tracking of Dendra2-labeled tumor cells through the Mammary Imaging Window is a technique which allows us to image the metastatic behavior of tumor cells in chosen tumor microenvironments over a timescale of days.

乳腺イメージングの窓からDendra2 Photoswitching

In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution1-4  has resulted in important insights into ...

Right Hemihepatectomy by Suprahilar Intrahepatic Transection of the Right Hemipedicle using a Vascular Stapler

Successful hepatic resection requires profound anatomical knowledge and delicate surgical technique. Hemihepatectomies are mostly performed after preparing the extrahepatic hilar structures within the hepatoduodenal ligament, even in benign tumours or liver metastasis.1-5. Regional extrahepatic lymphadenectomy is an oncological standard in hilar cholangiocarcinoma, intrahepatic cholangio-cellular carcinoma and hepatocellular carcinoma, whereas lymph node metastases in the hepatic hilus in patients with liver metastasis are rarely occult. Major disadvantages of these procedures are the complex preparation of the hilus with the risk of injuring contralateral structures and the possibility of bleeding from portal vein side-branches or impaired perfusion of bile ducts. We developed a technique of right hemihepatectomy or resection of the left lateral segments with intrahepatic transection of the pedicle that leaves the hepatoduodenal ligament completely untouched. 6 However, if intraoperative visualization or palpation of the ligament is suspicious for tumor infiltration or lymph node metastasis, the hilus should be explored and a lymphadenectomy performed.

This video describes a right hemihepatectomy with intrahepatic transection of the right hemipedicle leaving the hepatoduodenal ligament completely untouched.

血管ホッチキスを使って右HemipedicleのSuprahilar肝内切除により右Hemihepatectomy

Successful hepatic resection requires profound anatomical knowledge and delicate surgical technique. Hemihepatectomies are mostly performed after ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development of a larger region of the embryo. This makes defining its role in a particular tissue difficult to analyze. For example, noggin overexpression in Xenopus laevis embryos causes the expansion of the entire anterior region, including the eye1,2. From this result, it is not known if Noggin plays a direct role in eye determination or that by causing an expansion of neural tissue, Noggin indirectly affects eye formation. Having this complex phenotype makes studying its eye-specific role in cell fate determination difficult to analyze. We have developed an assay that overcomes this problem. Taking advantage of the pluripotent nature of the Xenopus laevis animal cap 3, we have developed an assay to test the ability of gene product(s), like noggin or the eye field transcription factors (EFTFs), to transform caps into particular tissue or cell types by transplanting this tissue onto the side of the embryo 4. While we have found either Noggin protein treatment or a collection of transcription factors can determine retinal cell fate in animal caps, this procedure could be used to identify gene product(s) involved in specifying other tissues as well.

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.

でアニマルキャップの移植（ACT）アッセイを用いて組織の決定アフリカツメガエル</em

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

三次元混合細胞スフェロイド共培養から乳腺上皮細胞の分離

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA精製によってクロマチンの単離（チャープ）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

髪の再生の間に上皮間葉シグナル伝達の迅速遺伝子解析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

マウス結腸粘膜の顕微解剖のための凍結切片法

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

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.