These studies were funded by CIHR and CBCRA grants to DWL. IP was funded by fellowships from CIHR-STP, FRSQ and CIHR. MKGS was funded by OGS and CIHR-STP scholarships. The authors thank Kevin Barr for his assistance with mouse breeding.

1. Mammary gland dissection 
<ol>
<li>Euthanize the mouse using CO2 inhalation. Avoid cervical
dislocation if possible since it may damage major blood vessels in the
neck and result in accumulation of blood around mammary glands,
rendering the dissection more difficult. However, if other criteria
are to be evaluated in the same mouse, such as blood levels of
oxytocin, alternative euthanasia methods may be necessary, though this
must be evaluated by your IACUC.</li>
<li>On a polystyrene foam wrapped in foil, place the mouse on its back and spread the four limbs. Pin firmly all four limbs using 16 to 20 gauge needles (Figure 1A).</li>
<li>Spray the mouse generously with 70% ethanol.</li>
<li>Using forceps, pull up the abdominal skin at the midline and make a small incision with sharp scissors.</li>
<li>Starting from the incision, cut the skin up to the neck of the animal while avoiding puncturing the abdominal or thoracic cavities (Figure 1B). </li>
<li>Cut the skin on the front legs to the midline incision, resulting in a &quot;Y shape&quot; (Figure 1B). Cut the skin on the rear limbs the same way (Figure 1B). </li>
<li>Using hemostatic forceps, gently peel the abdominal and thoracic skin to the side of the mouse. If needed, gently cut conjunctive tissue.</li>
<li>Fix the skin to the polystyrene foam using 16 to 20 gauge needles, exposing the mammary glands attached to the underside of the skin. Proceed and complete one side at a time (Figure 1C).</li>
<li>Using forceps, gently lift the mammary gland of interest and cut between the gland and the skin with small sharp scissors, starting from the outside towards the spine of the animal. Make sure you cut carefully, so no pieces of skin or muscle remain on the gland. All mammary glands (Figure 1) can be removed using this protocol, but abdominal mammary glands (#4) are best for whole mounting. The 2nd and 3rd glands have some interdigitated muscles and while they can be used for whole mount and histology, they might not be appropriate for all analyses.</li>
</ol>
2. Whole mount 
<ol>
<li>At least one day prior to dissection, prepare carmine alum solution by dissolving 1g of carmine and 2.5g of aluminum potassium sulfate in 500 ml of distilled water and boil for 20 minutes in a 1 liter Erlenmeyer flask. Adjust the final volume to 500 ml with water. Filter through a Whatman paper and add a small amount of thymol (few grains) as a preservative. Refrigerate. After staining, carmine alum solution can be poured back to the stock bottle and reused many times over several weeks. Make a fresh solution when color becomes faint. </li>
<li>After excising the mammary gland from the mouse (step 1), spread it directly on a glass slide. Try to flatten it as much as possible, keeping the original in situ shape. The gland will stick on the slide nicely when properly stretched.</li>
<li>Fix the gland by immersing it, on the slide, in Carnoy's fixative (100% EtOH, chloroform, glacial acetic acid; 6:3:1) for 4 hours in the fume hood at room temperature in a jar. Note that others have used 2 hours which seems to be sufficient. Alternatively, glands can also be fixed at 4&deg;C overnight.</li>
<li>Wash in 70% ethanol for 15 min. Then, rehydrate gradually in water by removing half of the ethanol solution from the jar and replacing it with ddH2O. Incubate for 5 min. Repeat 3 times. Alternatively, use 70%-, 35%-, 15%- ethanol and water baths for 5 minutes each.</li>
<li>Rinse in distilled water for 5 minutes. </li>
<li>Stain in carmine alum overnight at room temperature. It should be noted that staining might take longer depending on the thickness of the gland. </li>
<li>Remove carmine alum stain to reuse and gradually dehydrate stained tissue through serial ethanol baths (50%, 70%, 95%, 100%) for 5 min each and clear in xylene, or in a non-toxic alternative such as Histo-clear, overnight. Both dehydration and clearing might also require longer incubations with thicker samples.</li>
<li>Immerse mammary gland in methyl salicylate. Mammary glands can be kept in methyl salicylate in a fume hood until images are ready to be taken.</li>
<li>Images can be taken in a fume hood with a regular digital camera positioned on a tripod (use &quot;macro&quot; function if available), by placing the slides on a light table. Use a dissecting microscope for higher magnification. Alternatively, mammary glands can be mounted in media such as Permount (after step 2.7) allowing for pictures to be taken outside a chemical fume hood on any imaging platform providing the mounts are sufficiently thin.</li>
<li>In order to quantify mammary gland development using whole mount, measurements such as the number of ramifications (or side-branches) along portions of the ducts, the length of the primary ducts, or a ratio of epithelial to adipose tissue area could be assessed. Finally, after photographic documentation, mammary gland whole mount can be embedded in paraffin for sectioning and conventional histological staining.</li>
</ol>
3. Oxytocin-induced milk ejection
<ol>
<li>Separate the pups from the dam just after they have been fed. Wait 1 hour before performing the assay.</li>
<li>Sacrifice the mother and expose the mammary glands as described before (steps 1.1 to 1.8), without removing them from the animal.</li>
<li>Start the assay by evenly dropping either PBS or the oxytocin solution directly on the mammary gland of interest, using a transfer pipette. A large spectrum of doses can be used; &tilde;8 pg/ml (physiological dose) is typically used. However, high non-physiological doses (up to 1 mg/ml) can be used to ensure the absence of response in genetically modified mouse models. For this procedure, it is recommended to use thoracic (#2-3) glands, using one side as a control (exposed to PBS) and the other side to test milk ejection (oxytocin exposure).</li>
<li>Incubate for 1 minute and remove the solution by carefully aspirating it with a transfer pipette. Milk entry into the ducts can be monitored by video tape recording, or by taking images before and after PBS or oxytocin exposure.</li>
</ol>
4. Representative Results: 
In a good whole mount, the mammary gland is nicely spread and the epithelium ducts are easily observable (Figure 2A). If the gland is not well-spread, it can lift up in part or totally from the slide when placed in the fixative solution. The gland will then become hard and unusable (Figure 2B). Similarly, if the gland is not well dissected, either some part of the epithelium can be missing (Figure 2C) or remaining parts of the skin or abdominal muscle will interfere with the analysis of the gland (Figure 2D).
For the oxytocin assay, it is important to separate the suckling pups from the dam for the same amount of time on every occasion. In this way, you will make sure that no milk or small amounts of milk is present in the ducts prior to oxytocin exposure, but also that enough milk is present in the alveoli that can be ejected into the ducts by myoepithelial contractions (Figure 3A). If pups are removed too soon, milk can accumulate in the alveoli and in the ducts rendering the effect of oxytocin difficult to monitor (Figure 3B). Alternatively, if the experiment is performed too early after pup removal, milk won't have time to accumulate in the alveoli and oxytocin-induced contractions of myoepithelial cells won't trigger enough milk release into the ducts to be observed (Figure 3C).
<img src="/files/ftp_upload/2828/2828fig1.jpg" alt="Figure 1"/> 
Figure 1. Mouse mammary gland dissection. Pin the mouse on its back by the four limbs (A). Cut the skin first from the belly to the neck. Then make perpendicular incisions from the center of the belly to each limb, as shown by the dotted line (B). Gently peel the skin on the side of the animal and pin it, exposing the mammary glands (C).
<img src="/files/ftp_upload/2828/2828fig2.jpg" alt="Figure 2"/> 
Figure 2. Mouse mammary gland whole mount. Epithelium ducts (arrows) and the lymph node (arrowhead) are easily observed in a nicely spread mammary gland whole mount of gland #4 (A). A poorly spread mammary gland may become detached from the slide and collapsed, rendering it unusable (B). An improperly excised mammary gland can either have missing parts (C) or pieces of muscle or skin remaining (D). Note that the 2nd and 3rd glands have interdigitated muscle, but can still be used for whole mount or histology.
<img src="/files/ftp_upload/2828/2828fig3.jpg" alt="Figure 3"/> 
Figure 3. Oxytocin-induced milk ejection can be observed in epithelium ducts (arrows) following oxytocin exposure (A). However, if ducts are already filled with milk (arrows) at the beginning of the assay due to an inappropriately long period of latency after the last suckling of the pups, further accumulation of milk in the ducts (arrows) after oxytocin exposure is hard to observe (B). Alternatively, a short latency period after pup suckling will not allow for enough accumulation of milk into the alveoli to be properly observed in the ducts (arrows) after oxytocin exposure (C). Images were taken before and after oxytocin exposure using a numeric camera (Cybershot, Sony).
<img src="/files/ftp_upload/2828/2828fig4.jpg" alt="Figure 4"/> 
Figure 4. Schematic representations of whole mounting and oxytocin treatment assays.

<ol>
	<li>Geddes, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inside+the+lactating+breast:+the+latest+anatomy+research.">Inside the lactating breast: the latest anatomy research.</a> J Midwifery Womens Health. 52, 556-556 (2007).</li><li>Sternlicht, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Key+stages+in+mammary+gland+development:+the+cues+that+regulate+ductal+branching+morphogenesis.">Key stages in mammary gland development: the cues that regulate ductal branching morphogenesis.</a> Breast Cancer Res. 8, 201-201 (2006).</li><li>Silberstein, G. B., Flanders, K. C., Roberts, A. B., Daniel, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+mammary+morphogenesis:+evidence+for+extracellular+matrix-mediated+inhibition+of+ductal+budding+by+transforming+growth+factor-beta+1.">Regulation of mammary morphogenesis: evidence for extracellular matrix-mediated inhibition of ductal budding by transforming growth factor-beta 1.</a> Dev Biol. 152, 354-354 (1992).</li><li>Daniel, C. W., Robinson, S., Silberstein, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+TGF-beta+in+patterning+and+growth+of+the+mammary+ductal+tree.">The role of TGF-beta in patterning and growth of the mammary ductal tree.</a> J Mammary Gland Biol Neoplasia. 1, 331-331 (1996).</li><li>Neville, M. C., Daniel, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mammary gland : development, regulation, and function. , (1987).</li><li>Oakes, S. R., Hilton, H. N., Ormandy, 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=The+alveolar+switch:+coordinating+the+proliferative+cues+and+cell+fate+decisions+that+drive+the+formation+of+lobuloalveoli+from+ductal+epithelium.">The alveolar switch: coordinating the proliferative cues and cell fate decisions that drive the formation of lobuloalveoli from ductal epithelium.</a> Breast Cancer Res. 8, 207-207 (2006).</li><li>Anderson, S. M., Rudolph, M. C., McManaman, J. L., Neville, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Key+stages+in+mammary+gland+development.+Secretory+activation+in+the+mammary+gland:+it's+not+just+about+milk+protein+synthesis!.">Key stages in mammary gland development. Secretory activation in the mammary gland: it's not just about milk protein synthesis!.</a> Breast Cancer Res. 9, 204-204 (2007).</li><li>Reversi, A., Cassoni, P., Chini, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxytocin+receptor+signaling+in+myoepithelial+and+cancer+cells.">Oxytocin receptor signaling in myoepithelial and cancer cells.</a> J Mammary Gland Biol Neoplasia. 10, 221-221 (2005).</li><li>Haslam, S. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+to+cell+interactions+and+normal+mammary+gland+function.">Cell to cell interactions and normal mammary gland function.</a> J Dairy Sci. 71, 2843-2843 (1988).</li><li>Plante, I., 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=Decreased+levels+of+connexin43+result+in+impaired+development+of+the+mammary+gland+in+a+mouse+model+of+oculodentodigital+dysplasia.">Decreased levels of connexin43 result in impaired development of the mammary gland in a mouse model of oculodentodigital dysplasia.</a> Dev Biol. 318, 312-312 (2008).</li><li>Talhouk, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterocellular+interaction+enhances+recruitment+of+alpha+and+beta-catenins+and+ZO-2+into+functional+gap-junction+complexes+and+induces+gap+junction-dependant+differentiation+of+mammary+epithelial+cells.">Heterocellular interaction enhances recruitment of alpha and beta-catenins and ZO-2 into functional gap-junction complexes and induces gap junction-dependant differentiation of mammary epithelial cells.</a> Exp Cell Res. 314, 3275-3275 (2008).</li><li>Palmer, C. A., Neville, M. C., Anderson, S. M., McManaman, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+lactation+defects+in+transgenic+mice.">Analysis of lactation defects in transgenic mice.</a> J Mammary Gland Biol Neoplasia. 11, 269-269 (2006).</li><li>Howlin, J., McBryan, J., Martin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pubertal+mammary+gland+development:+insights+from+mouse+models.">Pubertal mammary gland development: insights from mouse models.</a> J Mammary Gland Biol Neoplasia. 11, 283-283 (2006).</li><li>You, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+mammary+gland+development+in+prepubertal+male+rats+exposed+to+genistein+and+methoxychlor.">Modulation of mammary gland development in prepubertal male rats exposed to genistein and methoxychlor.</a> Toxicol Sci. 66, 216-216 (2002).</li><li>Grill, C. J., Cohick, W. S., Sherman, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postpubertal+development+of+the+rat+mammary+gland+is+preserved+during+iron+deficiency.">Postpubertal development of the rat mammary gland is preserved during iron deficiency.</a> J Nutr. 131, 1444-1444 (2001).</li><li>Hennighausen, L., Robinson, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Think+globally,+act+locally:+the+making+of+a+mouse+mammary+gland.">Think globally, act locally: the making of a mouse mammary gland.</a> Genes Dev. 12, 449-449 (1998).</li><li>Aupperlee, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-specific+differences+in+the+mechanisms+of+progesterone+regulation+of+murine+mammary+gland+development.">Strain-specific differences in the mechanisms of progesterone regulation of murine mammary gland development.</a> Endocrinology. 150, 1485-1485 (2009).</li><li>Montero Girard, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+estrogen+receptor-alpha+and+progesterone+receptor+A+expression+with+hormonal+mammary+carcinogenesis:+role+of+the+host+microenvironment.">Association of estrogen receptor-alpha and progesterone receptor A expression with hormonal mammary carcinogenesis: role of the host microenvironment.</a> Breast Cancer Res. 9, R22-R22 (2007).</li><li>Moore, D. M., Vogl, A. W., Baimbridge, K., Emerman, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+calcium+on+oxytocin-induced+contraction+of+mammary+gland+myoepithelium+as+visualized+by+NBD-phallacidin.">Effect of calcium on oxytocin-induced contraction of mammary gland myoepithelium as visualized by NBD-phallacidin.</a> J Cell Sci. 88, 563-563 (1987).</li></ol>

No conflicts of interest declared.

Mammary gland development and function are tightly orchestrated. Mutant mice may harbor gene mutations that can impair mammary gland development and function highlighting the need to evaluate gland architecture13,12. Whole mounting can be performed as described in this article at all stages of mammary gland development. Typically, development of the epithelium is assessed at the beginning of puberty ( &tilde;4 weeks old), in the middle of puberty ( &tilde;6 weeks old) and after puberty ( &tilde; 10-12 weeks ol...

<table border="1">
	<tbody>
 	<tr>
 	<th>Reagent</th>
 <th>Company</th>
 <th>Catalogue number</th> 
 </tr>
 <tr>
 	<td>Carmine</td>
 <td>Sigma-Aldrich</td>
 <td>C1022</td> 
 </tr>
 <tr>
 	<td>Aluminum potassium sulfate</td>
 <td>Sigma-Aldrich</td>
 <td>A6435</td> 
 </tr>

 <tr>
 	<td>Thymol</td>
 <td>Sigma-Aldrich</td>
 <td>T0501</td> 
 </tr>
 <tr>
 	<td>Methyl salicylate</td>
 <td>Sigma-Aldrich</td>
 <td>M6752</td> 
 </tr>
 <tr>
 	<td>Oxytocin</td>
 <td>Sigma-Aldrich</td>
 <td>O3251</td>
 </tr> 
 </tbody>
</table>

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.

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

Evaluation of Mammary Gland Development and Function in Mouse Models

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.

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

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

Research

JoVE Journal

Biology

37.0K Views.  University of Western Ontario. 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.

Video: Evaluation of Mammary Gland Development and Function in Mouse Models

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

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

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

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

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

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 ...

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

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

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

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

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

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland undergoes extensive modeling and re-modeling, alter susceptibility to carcinogen-induced mammary tumors. Similar findings have been described in humans: for example, high birthweight increases later risk of developing breast cancer, and dietary intake of soy during childhood decreases breast cancer risk. It is thought that these prenatal and postnatal dietary modifications induce persistent morphological changes in the mammary gland that in turn modify breast cancer risk later in life. These morphological changes likely reflect epigenetic modifications, such as changes in DNA methylation, histones and miRNA expression that then affect gene transcription . In this article we describe how changes in mammary gland morphology can predict mammary cancer risk in rats. Our protocol specifically describes how to dissect and remove the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology according to three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use the data to predict risk of developing mammary cancer.

Our protocol describes how to dissect the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology using three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use these results to predict mammary cancer risk in rats which were exposed to dietary modifications in utero or during prepuberty.

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 ...

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

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

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

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

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

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

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

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

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

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

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

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

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

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish5. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria &#8216;dipstick&#8217;, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function1,6-9.

The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD.

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...

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 ...

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.

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 ...