1。移植的上皮细胞的制备可以移植到新鲜或冷冻的捐助上皮组织清除的脂肪垫，但事先准备的冷冻捐助上皮在移植效率的最小下降（&gt; 85％的效率）更方便。编制冻结​​捐助上皮协议如下： 准备捐助移植上皮之前，准备冻结上皮和有关分装到冷冻管1毫升媒体。这可以保存在4 ° C，直到需要长达6个月。冻结媒体包含与HEPES和碳酸氢钠，10％胎牛血清（FBS）和10％二甲基亚砜（DMSO）3缓冲的DMEM/F12媒体。 收获从最近安乐死的雌性小鼠乳腺上皮细胞。 <ol><li>鼠标用70％乙醇洗涤。 </li><li>通过从耻骨至胸骨体延伸到墙上，没有刺耳的腹侧体腔浆膜膜中旬矢状切。从最初向每个后腿（图1）耻骨切口作出的斜剪。 </li><li>去皮，浆膜膜暴露5 日腹股沟乳腺筋膜附着在体壁的下部离体壁。最好的方式实现这一目标是通过抓一双弯钳弯曲部分的浆液膜仔细，小心不要刺破腹体腔。同时，掌握直锯齿钳包膜切缘和离体壁拉浆液膜。 </li><li>识别乳腺内的淋巴结。淋巴结是位于在乳腺中的三个突出的血管的交点，可以感受到作为密集的结节内脂肪垫（图2）。一双眼睛剪刀，NIP节点周围的脂肪组织的一角。随着手指上的皮肤表皮中脱颖而出，适用于压力，提高节点出的脂肪垫和一双弯钳捏。丢弃的淋巴结。 </li><li>虽然皮肤的边缘，掌握乳腺脂肪垫一双弯钳的弧形边缘，并逐步剥离脂肪垫离体壁。按照对背水面的鼠标，为您删除在一块整个乳腺脂肪垫的方向。 </li><li>放在一个无菌塑料切割块的乳腺。添加少量的媒体，约200μL，以保持湿润的腺体表面。与一个新的刀片，切碎成1毫米件的腺体。分成四个部分碎腺体和4个冷冻管钳冻结媒体之间分配。放在一个NALGENE“先生雾细胞冷冻异丙醇250毫升外舱的离心管。NALGENE冷冻在-70 ° C冰箱24小时。乳腺片段储存在液氮冷冻小瓶，直到所需的每个移植。离心管会包含足够的乳腺片段移植到清除4日至8宿主小鼠的脂肪垫。 </li></ol> 2。移植细胞的制备我们的实验室已移植了不同的细胞类型，包括永生化乳腺上皮细胞系，乳腺肿瘤细胞株或成鼠标的主要乳腺细胞清除脂肪垫。逗号- D细胞移植的准备协议如下： <ol><li>逗号- D细胞培养定期MECL媒体的DMEM：F12与2％的成年牛血清（ABS）mEGF，胰岛素，抗生素/ Antimycotic（AB / AM）补充。 </li><li>收获逗号D细胞，当细胞85％-90％汇合。 </li><li>在1200RPM离心5分钟沉淀细胞的细胞悬液。新鲜MECL媒体重悬细胞，调整细胞浓度为5 × 10 6 /毫升。 </li></ol>对于原发性乳腺细胞的移植，主要的乳腺上皮细胞是从小鼠乳腺5日通过机械和酶解分离。细胞是悬浮于DMEM：F12媒体和调整到所需的浓度，一般为5 × 10 6 /毫升。 3。清除小鼠乳腺脂肪垫的标准程序<ol><li>麻醉3个星期的旧主机的鼠标。选择&lt;12克，以确保尚未启动，乳腺导管增长的老鼠。我们使用经口200mg/kg体重2,2,2 - tribromoethanol。 </li><li>鼠标麻醉后，坚决抑制鼠标，腹侧，四肢伸开在手术阶段，使腹部是嘲讽。手术前，管理止痛药;布比卡因（&lt;1mg/kg）或丁丙诺啡（0.05mg/kg），皮下注射。 </li><li>把握几个毫米以上骨盆钳皮肤和解除它从腹部<html> 。聂皮肤用剪刀，并通过一个中期矢状切开从耻骨至胸骨体延伸到墙上。不要刺破腹体腔浆膜膜。不要让您的初始切口太靠近尿道，使伤口剪辑不会干扰鼠标的能力，小便。 </li><li>从最初的切口，通过皮肤斜削减从中线整个骨盆对每个后腿。 </li><li>使用弧形面积弯钳轻轻抓住浆液膜。用组织钳把握的皮肤切缘拉体壁远离浆液膜和揭露5 个位于体壁的下部的筋膜腹股沟乳腺。 </li><li>烧灼乳头，节点和两个动脉导致的节点（图2） </li><li>第四和第五腺分开。烧灼第五腺的保证金，以防止从6日腺体长入。 </li><li>除以整个脂肪垫只是腹节点（对乳头侧）用剪刀切割第四乳腺脂肪垫。用弯钳，牢牢把握腹脂肪垫部分拉在一块的体壁。按腹部分的两个幻灯片之间的脂肪垫和玻璃组织学染色包含卡诺的固定液菜。该组织将与胭脂红明矾溶液后固定染色检查清除边缘（图3）。清洁弯钳乳腺脂肪垫去除所有痕迹的删除部分地区周围的筋膜。 </li></ol> 4。另类“饶程序清除脂肪垫后已经成为一个标准程序清除小鼠乳腺脂肪垫主管，他们可能更愿意尝试创伤小饶程序开发鲽鱼等 （2008）2。 <ol><li>准备为标准的程序鼠标。 </li><li>抓住第四腹股沟乳腺乳头和乳头周围用剪刀的尖端切一个3-5毫米的圆周圈。 </li><li>拉出乳头，远离身体的皮肤中分离的切割部分和第四腹股沟乳腺孔通过绘制。 </li><li>识别节点。使用电凝烧灼的节点，并通过孔的第 6 次乳腺。 </li><li>剪切和分离的节点之前的5 次乳腺最浅表的部分。乳头，皮肤和乳腺脂肪垫含有上皮部分的圆形区域，在一块被删除。 </li></ol> 5。组织/细胞移植<ol><li>移植的上皮细胞碎片，除霜一个离心管上皮细胞和上皮碎片转移到培养皿中含有新鲜的媒体，以稀释的二甲基亚砜。制作了一个小洞，在清理用剪刀尖新的上皮细胞的脂肪垫或放在口袋里。放入口袋针钳的捐助上皮1毫米一块。握住组织钳封闭你退出针钳，组织到位，的口袋。 </li><li>移植细胞，注入10μL媒体的50,000个单元格，使用汉密尔顿一个50μL注射器用22号针头到每个脂肪垫。 </li></ol> 6。关闭/鼠标复兴<ol><li>排列闭幕皮肤。抓住反对皮肤的利润率，保持他们一起用钳，电梯从腹侧体腔浆膜膜他们离开，并关闭伤口剪辑。通常情况下，有必要使用两个剪辑接近中期矢状切口和一个剪辑的标准程序的每一个斜切口。避免妨碍尿道。这可能是必要关闭在皮肤上，在中期矢状位和斜切口缝合的交集的差距。不遗余力程序将只需要一个每边的伤口剪辑。 </li><li> 。鼠标放置在笼子里，并保持在一个加热灯笼，直到鼠标复苏。 </li><li> 2周后应去掉伤口剪辑。 </li></ol><img src="/files/ftp_upload/1849/1849fig1.jpg" alt="图1" /> 图1。切口模式的示范。 <img src="/files/ftp_upload/1849/1849fig2.jpg" alt="图2" /> 图2。淋巴结，血管，乳头和内源性上皮细胞的位置。烧灼位置表示。 <img src="/files/ftp_upload/1849/1849fig3.jpg" alt="图3" /> 图3。整装表明，内源性上皮内圆的面积限制，因此利润率“明确”。 7。代表性的成果_r.jpg“ALT =”图4“/&gt; 图4。整装3周龄鼠标显示清除利润率从脂肪垫。 <img src="/files/ftp_upload/1849/1849fig2_r.jpg" alt="图5" /> 图5。整装从10周龄乳腺上皮内源性清除脂肪垫鼠标。 <img src="/files/ftp_upload/1849/1849fig3_r.jpg" alt="图6" /> 图6。苏木精和曙红染色部分清除从10周龄小鼠的脂肪垫。 <img src="/files/ftp_upload/1849/1849fig4_r.jpg" alt="图7" /> 图7：从10周龄正常乳腺导管树移植上皮鼠标整装。 <img src="/files/ftp_upload/1849/1849fig5_r.jpg" alt="图8" /> 图8。苏木精和曙红染色部分移植上皮开发的乳腺导管。 <img src="/files/ftp_upload/1849/1849fig6_r.jpg" alt="图9" /> 图9。苏木精和伊红染色部分移植上皮开发出了乳腺肿瘤。

<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>

在这份报告中，我们提出两个替代方法，结算准备乳腺上皮移植在小鼠乳腺脂肪垫。已在使用的标准程序，自1959年发展DeOme等。1虽然更具侵入性的，标准的方法是培训新手的首选方法，因为乳腺的结构和方向很容易观看。然而，饶程序可能会首选由更有经验的外科医生， 因为几个优点2。最重要的是，不遗余力程序是创伤小，需要更短的时间内，可以使用吸入麻醉的短暂时...

mammary epithelial transplant procedure

本文介绍和比较脂肪垫DeOme KB 等开发的清关程序 。1和备用程序开发布里尔 B 等 。2，乳腺上皮移植手术。乳腺移植手术被广泛应用于乳腺生物学家，因为它需要的事实，乳腺上皮细胞的显着发展，直到进入青春期后，不会发生的优势。在3周龄，乳腺上皮树的生长是局限于乳头附近，脂肪垫主要是缺乏对乳腺上皮细胞，但7周龄的乳腺导管上皮树延伸整个脂肪垫。因此，如果这种含有上皮细胞的脂肪垫一小部分，地区之间的乳头和淋巴结，是在3周拆除，内源性上皮永远不会填充乳腺脂肪垫，脂肪垫是描述为“清除“。这时，从其他来源的乳腺上皮可在清除脂肪垫，它有可能延长到了脂肪垫乳腺导管树木移植。利用这个程序已经在许多的实验模型，包括在转基因乳腺上皮组织的肿瘤表型无基因型的3对整个动物的混杂影响考试，在鉴别乳腺干细胞移植细胞有限稀释4,5的，确定增生性结节，如果进行乳腺肿瘤6，评估前激素对乳腺上皮细胞7,8的行为暴露的影响。 三个星期的旧主机小鼠麻醉，在手术阶段清理和克制。一个中期矢状切口，通过皮肤，但没有腹膜，从耻骨扩展到胸骨。斜削减从整个骨盆向每条腿中旬矢状切口，通过皮肤。皮肤被拉到远离腹膜暴露第四腹股沟乳腺。脂肪垫是通过消除脂肪垫组织前淋巴结清除。上皮碎片或上皮细胞移植到其余的清除脂肪垫和鼠标是封闭的。

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

Mammary Epithelial Transplant Procedure

本文演示了由DeOme KB开发的程序<em&gt;等。</em&gt;（1959年）和备用程序开发布里尔乙<em&gt;等。</em&gt;（2008年）为结算第四腹股沟在乳腺片段，乳腺上皮细胞，或乳腺肿瘤细胞的移植准备柔毛小鼠乳腺脂肪垫。

乳腺上皮移植手术

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 次观看.  University of Massachussetts. 本文演示了由DeOme KB开发的程序等。（1959年）和备用程序开发布里尔乙等。（2008年）为结算第四腹股沟在乳腺片段，乳腺上皮细胞，或乳腺肿瘤细胞的移植准备柔毛小鼠乳腺脂肪垫。

视频: Mammary Epithelial Transplant Procedure

Investigating the Immunological Mechanisms Underlying Organ Transplant Rejection

调查的基本免疫机制，器官移植排斥反应

科研

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.

小鼠乳腺上皮细胞形成过程中Lactogenic分化的微球体

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.

通过乳腺成像窗口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.

右右Hemipedicle Suprahilar肝内血管吻合器横断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.

组织测定动物第移植（ACT）的含量非洲爪蟾</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.

从三维混合细胞球体共培养的乳腺上皮细胞的隔离

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纯化的染色质隔离（CHIRP）

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.

在毛发再生的上皮 - 间充质信号的快速遗传分析

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.

Cryosectioning方法小鼠结肠黏膜显微切割

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.