1. Preparation of Transplant Epithelium
Fresh or frozen donor epithelial tissue can be transplanted into the cleared fat pad, but prior preparation of frozen donor epithelium is more convenient with a minimal decrease in transplantation efficiency (&gt;85% efficiency). The protocol for the preparation of frozen donor epithelium is as follows:
Before preparing donor transplant epithelium, prepare freeze media for epithelium and aliquot about 1ml into cryovials. This can be stored at 4&deg;C for up to 6 months until needed. Freeze media contains DMEM/F12 media buffered with HEPES and NaHCO3, 10% fetal bovine serum (FBS) and 10% dimethyl sulphoxide (DMSO).
Harvest mammary epithelium from a recently euthanized female mouse.
<ol>
 <li>Wash the mouse with 70% EtOH.</li>
 <li>Make a mid-sagittal cut through the body wall extending from the pubis to the sternum without piercing the serous membranes of the ventral body cavity. Make an oblique cut from the initial incision at the pubis toward each hind leg (Figure 1).</li>
 <li>Peel the body wall away from the serous membranes to expose the 5th inguinal mammary gland attached to the fascia on the underside of the body wall. The best way to achieve this is by grasping the serous membranes carefully with the curved portion of a pair of curved forceps, being careful not to pierce the ventral body cavity. Simultaneously, grasp the cut edge of the integument with straight serrated forceps and pull the body wall away from the serous membranes.</li>
 <li>Identify the lymph node within the mammary gland. The lymph node is located at the intersection of three prominent blood vessels in the mammary gland and can be felt as a dense nodule within the fat pad (Figure 2). With the tip of a pair of eye scissors, nip the adipose tissue surrounding the node. With a fore finger on the epidermal side of the skin, apply pressure to raise the node out of the fat pad and pinch it off with a pair of curved forceps. Discard the lymph node.</li>
 <li> While holding the margins of the skin, grasp the mammary fat pad with the curved edge of a pair of curved forceps and gradually peel the fat pad away from the body wall. Follow the direction of the fat pad towards the dorsal surface of the mouse as you remove the entire mammary gland in one piece.</li>
 <li>Put the mammary gland on a sterile plastic cutting block. Add a small amount of media, about 200 &mu;l, to the surface of the gland to keep it moist. Mince the gland into 1mm pieces with a fresh razor blade. Divide the minced gland into four portions and distribute among four cryovials of freeze media with forceps. Put the cryovials in a Nalgene "Mr. Frosty cell freezer which has 250ml of isopropyl alcohol in the outer compartment. Put the Nalgene freezer in a -70&deg;C freezer for 24 hours. Store frozen vials of mammary fragments in liquid nitrogen until required for transplantation. Each cryovial will contain enough mammary gland fragments to transplant into the cleared fat pads of 4 to 8 host mice.</li>
</ol>
2. Preparation of Transplant Cells
Our lab has transplanted different cell types, including immortalized mammary epithelial cell lines, mammary tumor cell lines or primary mammary cells into a mouse cleared fat pad. The protocol for the preparation of Comma-D cells for transplantation is as follows:
<ol>
 <li>Comma-D cells are cultured in regular MECL media containing DMEM:F12 supplemented with 2% Adult Bovine Serum (ABS), mEGF, Insulin, Antibiotic/Antimycotic (AB/AM). </li>
 <li>Harvest Comma-D cells when the cells are 85%-90% confluent. </li>
 <li>Centrifuge the cell suspension at 1200rpm for 5 minutes to pellet the cells. Resuspend the cells in fresh MECL media and adjust the cell concentration to 5 x 106/ml. </li>
</ol>
For the transplantation of primary mammary cells, the primary mammary epithelial cells are isolated from mouse 5th mammary glands through mechanical and enzymatic dissociation. Cells are resuspended in DMEM:F12 media and adjusted to the desired concentration, usually 5 x 106/ml.
3. Standard Procedure for Clearing the Mouse Mammary Fat Pad
<ol>
 <li>Anesthetize the three week old host mouse. Select mice that are &lt;12 g to ensure that mammary ductal growth has not been initiated. We use 200mg/kg of body weight of 2,2,2-tribromoethanol. </li>
 <li>After the mouse is anesthetized, firmly restrain the mouse, ventral side up with limbs outstretched, on a surgical stage so that the abdomen is taunt. Prior to surgery, administer analgesics; either bupivacaine (&lt;1mg/kg) or buprenorphine (0.05mg/kg), subcutaneously.</li>
 <li>Grasp the skin with forceps a few millimeters above the pelvis and lift it up from the abdomen. Nip the skin with scissors and to make a mid-sagittal cut through the body wall extending from the pubis to the sternum. Do not pierce the serous membranes of the ventral body cavity. Do not make your initial incision too close to the urethra so that wound clips will not interfere with the mouse's ability to urinate.</li>
 <li>From the initial incision, make oblique cuts through the skin from the mid-line across the pelvis towards each hind leg.</li>
 <li>Use the curved area of the curved forceps to gently grasp the serous membranes. Use tissue forceps to grasp the cut margin of the skin and pull the body wall away from the serous membranes and expose the 5th inguinal mammary gland located on the fascia on the underside of the body wall.</li>
 <li>Cauterize the nipple, node and two arteries leading to the node (Figure 2)</li>
 <li>Separate the fourth and fifth glands. Cauterize the margin of the fifth gland to prevent ingrowth from the 6th gland.</li>
 <li>Divide the fat pad of the fourth mammary gland by cutting across the fat pad with scissors just ventral (side towards nipple) of the node. Use curved forceps to firmly grasp the ventral portion of the fat pad and pull it away from the body wall in one piece. Press the ventral portion of the fat pad between two slides and put in a glass histology staining dish containing Carnoy's fixative. This tissue will be fixed and stained later with carmine alum solution to check for cleared margins (Figure 3). Clean the fascia around the region of the removed portion of the fat pad to remove all traces of the mammary gland with the curved forceps. </li>
</ol>
4. Alternative "Sparing Procedure for Clearing the Fat Pad
After one has become competent with the standard procedure for clearing the mouse mammary fat pad, they may prefer to attempt the less invasive sparing procedure developed by Brill et al. (2008)2.
<ol>
 <li>Prepare the mouse as for the standard procedure.</li>
 <li>Grasp the nipple of the fourth inguinal mammary gland and cut a 3-5 mm circumference circle around the nipple with the tip of scissors.</li>
 <li>Pull the nipple away from the body to detach the cut portion of skin and draw the fourth inguinal mammary gland out through the hole.</li>
 <li>Identify the node. Use the cautery to cauterize the node and the 6th mammary gland through the hole.</li>
 <li>Cut and detach the most superficial portion of the 5th mammary gland just before the node. The nipple, circular area of skin and portion of the mammary fat pad containing epithelium are removed in one piece. </li>
</ol>
5. Tissue/Cell Transplantation
<ol>
 <li>For transplanting epithelium fragments, defrost a cryovial of epithelium and transfer the epithelial fragments to a Petri dish containing fresh media to dilute out the DMSO. Make a small hole or pocket in the cleared fat pad with scissor tip for the new epithelium. Put a 1mm piece of donor epithelium into the pocket with needle forceps. Hold the pocket closed with tissue forceps as you withdraw the needle forceps so the tissue remains in place.</li>
 <li>For transplanting cells, inject 50,000 cells in 10 &mu;l of media into each fat pad using a 50&mu;l Hamilton syringe with a 22s gauge needle. </li>
</ol>
6. Closing/Revival of Mouse
<ol>
 <li>Arrange the skin for closing. Grasp opposing margins of skin, hold them together with forceps, lift them away from the serous membranes of the ventral body cavity and close with wound clips. Typically, it is necessary to use two clips to close the mid-sagittal incision and one clip for each oblique incision for the standard procedure. Avoid obstructing the urethra. It may be necessary to close gaps in the skin at the intersection of the mid-sagittal and oblique incisions with a suture. The sparing procedure will only require one wound clip for each side.</li>
 <li>. Place the mouse in her cage and keep the cage under a heating lamp until the mouse has revived. </li>
 <li>The wound clips should be removed after 2 weeks.</li>
</ol>
<img src="/files/ftp_upload/1849/1849fig1.jpg" alt="Figure 1" /> 
 Figure 1. Demonstration of incision pattern.
<img src="/files/ftp_upload/1849/1849fig2.jpg" alt="Figure 2" /> 
 Figure 2. Location of lymph node, blood vessels, nipple and endogenous epithelium. Cauterize at locations indicated.
<img src="/files/ftp_upload/1849/1849fig3.jpg" alt="Figure 3" /> 
 Figure 3. Whole mount demonstrates that endogenous epithelium is restricted to the area within the circle and therefore margins are "clear'.
7. Representative Results
<img src="/files/ftp_upload/1849/1849fig1_r.jpg" alt="Figure 4" /> 
 Figure 4. Whole mount of removed fat pad from 3 week old mouse showing cleared margins.
<img src="/files/ftp_upload/1849/1849fig2_r.jpg" alt="Figure 5" /> 
 Figure 5. Whole mount from a 10 week old mouse showing fat pad cleared of endogenous mammary epithelium.
<img src="/files/ftp_upload/1849/1849fig3_r.jpg" alt="Figure 6" /> 
 Figure 6. Hematoxylin and eosin stained section of a cleared fat pad from a 10 week old mouse.
<img src="/files/ftp_upload/1849/1849fig4_r.jpg" alt="Figure 7" /> 
 Figure 7. Whole mount from a 10 week old mouse showing normal mammary ductal tree developed from transplanted epithelium.
<img src="/files/ftp_upload/1849/1849fig5_r.jpg" alt="Figure 8" /> 
 Figure 8. Hematoxylin and eosin stained section of mammary ducts developed from transplanted epithelium.
<img src="/files/ftp_upload/1849/1849fig6_r.jpg" alt="Figure 9" /> 
 Figure 9. Hematoxylin and eosin stained section of a mammary tumor developed from transplanted epithelium.

<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>

In this report, we present two alternative methods for clearing the mouse mammary fat pad in preparation for mammary epithelial transplants. The standard procedure has been in use since its development in 1959 by DeOme et al.1 While more invasive, the standard method is the preferred method for training the novice because the structure and orientation of the mammary gland are readily viewed. However, the sparing procedure may be preferred by the more experienced surgeon because of several advantages<s...

mammary epithelial transplant procedure

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, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

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

Mammary Epithelial Transplant Procedure

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

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, ...

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30.9K vistas.  University of Massachussetts. En este artículo se muestra el procedimiento desarrollado por Enviaselo KB Et al. (1959) y el procedimiento que preserve desarrollado por Brill B Et al. (2008) para limpiar el cuarto inguinal almohadilla de grasa mamaria de un ratón de la pubertad en la preparación para el trasplante de fragmentos de mama, las células epiteliales mamarias, o las células mamarias tumorales.

Video: Mammary Epithelial Transplant Procedure

Investigating the Immunological Mechanisms Underlying Organ Transplant Rejection

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.

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.

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.

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

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.

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.

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.

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.

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

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.

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

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.