Yazarlar hayvanlar ile ona yardım için stabilize SiRNA çözüm ve Heather Tobin hazırlamak için Michael Goldberg teşekkür etmek istiyorum. Bu çalışma DEI DOD Yenilikçi Ödülü W81XWH-08-1-0659 tarafından finanse edildi. SK doktora sonrası eğitim Susan G Komen vakıf (KG101329) tarafından desteklenmiştir. Yazarlar şematik hazırlanması için Kristin Johnson teşekkür etmek istiyorum.

1.. Enjeksiyon solüsyonunun hazırlanması <ol><li> Fosfat tamponlu tuzlu su (PBS) ya da ilgili diğer bir sulu çözelti içinde% 0.2 Evans mavi boya hazırlayın. Bu vital boya intraduktal enjeksiyon başarısını değerlendirmek için yararlı olan ve tekniğin uzmanlığı geliştirirken bir görüntüleme aracı olarak önerilir. </li></ol> Gerekli, enjekte edilecek hacmi bezlerinin sayısı ve konumu bağlıdır. Dişi farelere her 10 meme bezleri, enjekte edilebilir, ancak nedeniyle daha küçük boyuta bezi, bez çiftleri 1 ve 5, tipik olarak çözeltinin 10 ul enjekte edilir. Diğer tüm meme çiftleri 20 ul enjekte edilir. Bu birimler tüm duktal ağacı doldurmak için yeterlidir. 2. Ameliyat öncesi hazırlık <ol><li> Her bir farenin vücut ağırlığını kaydedin. Tüm klinik öncesi çalışmalarda olduğu gibi, hayvan ağırlıkları potansiyel toksisitesini değerlendirmek üzere (haftada iki kez ya da daha fazla) düzenli olarak izlenmelidir. </li><li> M uyutmakizofluran odacığı kullanılarak ouse ve göz yağ uygulanır. İşlem sırasında fareler, bir burun konisiyle oksijen izofluran inhale% 2-4 konsantrasyon kullanılarak anestezi devam edecektir. Dikkatle buna izofluran seviyesini ayarlayarak, solunum hızında değişiklikler için fareyi izlemek. </li><li> Deri altından önce analjezi gibi prosedüre meloksikam (5-10 mg / kg) enjekte edilir. </li><li> Meme alan bir over-the-counter epilasyon kremi uygulayın. 5 dakika bekleyin ve hafifçe dairesel hareketlerle bir pamuk uçlu aplikatör ile gevşek saç kaldırmak. Ilık su ile ıslatılmış nemli bir kağıt havlu kullanarak krem ​​çıkarın. Tıraş nedeniyle meme zarar verme riskine tavsiye edilmez. </li><li> Yavaşça aşağı ekstremitelerde taping tarafından Stereoskop altında fareyi sabitleyin. </li><li> Alkol bezlerden ile temiz enjeksiyon siteleri. </li></ol> 3. İntraduktal enjeksiyon <ol><li> Stereoskop altında enjekte edilmesi uygun meme bulun. Tuhaf mikro diseksiyon ince kullanınmeme açılmasını kapsayan herhangi bir ölü deri kaldırmak için zers. </li><li> Sabitlenmiş bir 33 G iğne ile merkezi metal bir 50 ul şırınga içine yükleyin enjeksiyon solüsyonu 10-20 ul. Bazen, enjeksiyondan sonra, az miktarda (1 ul ya da daha az) iğne çekerek üzerine meme dışarı sızıntı olabilir. Bu nedenle, potansiyel kaybı hesaba katmak için sırasıyla 11 veya 21 ul enjekte etmek için tavsiye edilir. </li><li> Ince cımbız ile hafifçe meme tutun ve enjeksiyon için konumlandırmak için hafifçe kaldırın. Bu meme kesmek için gerekli değildir. </li><li> Hızla duktal lümen içindeki sıvıyı hareket nedeniyle potansiyel hasarı en aza indirmek için yavaş yavaş çözüm enjekte edilir. Enjeksiyon oranı, yaklaşık olarak 40 | il / dk &#39;da muhafaza edilmelidir. </li></ol> 4. Ameliyat Sonrası Bakım <ol><li> Enjeksiyon yerinde gözlemlemek. Meme bölge veya çevre dokuya travma izi yok olmalıdır. Meme çevresindeki bölgede şişlik olası bir meme yağ pedi enjeksiyonu rathe gösterirBaşarılı bir intraduktal enjeksiyon daha r. </li><li> Burun konisi hayvan çıkarın ve kurtarma için ayrı bir kafeste taşımak. Hipotermi önlemek ve kurtarma yardımcı olmak için bir ısı lambası altında kafes yerleştirin. Fareler tek başına ev sahipliği ve onlar bilinci ve hareketlilik yeniden kadar yakından takip edilecektir. </li></ol> 5. Meme Bezi Doku Analizi <ol><li> Çalışmanın tamamlanmasından sonra, fareler bir izolasyon odası içinde CO2 sıkıştırılmış gaz, aşağıdaki servikal dislokasyon ile ötenazi vardır. Meme bezleri kesilir ve bütün montaj hazırlanması 11, histoloji, ya da RNA ve protein izolasyonu için kullanılabilmektedir. </li></ol>

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

Yazarlar finansal açıklamaları var.

Transgenik ve nakavt farenin göğüs kanserinde tek tek genlerin in vivo rolünü incelemek için çok değerli araçlardır. Bununla birlikte, pahalı ve zaman alıcı, bu hayvanlar oluşturmak için olan ve gen yok etme bazen meme bezi üzerinde bu genin özel etkileri fark bize önleyen bu genellikle yaygındır. Bu nedenle, hedef knockdown diğer organlarda spesifik olmayan yan etkiler ve toksisite sorunları giderecektir. Burada sunulan siRNA intraduktal enjeksiyon fare meme bezi lokalize gen demonte sağlar. Bu teslimat siRNA ile sınırlı değildir meme bezi ile bir hedef ve lokalize ilaç verilmesi için hızlı bir yöntemdir. Bu gibi bir meme bezi özel ilaç verme diğer organlara spesifik olmayan yan etkiler ve toksisite önler ve meme bezi gelişimi ve göğüs tümör oluşumu sırasında belirli zaman noktalarında gen değişikliklerinin çalışma sağlar. Si hiçbir kanıt yokturOlmayan enjekte bezlerinde RNA parçacıklar, karaciğer ya da bu yöntem meme bezi lokalize gen demonte için tercih edilen bir yol sunar daha fazla kanıt temin etkilenebilir. Ayrıca, intraduktal enjeksiyon terapötik maddelerin uzun süreli izlenmesi için izin vermek için birkaç ay boyunca haftada bir veya iki haftada bir tekrarlanabilir. Bu teknik, fare meme kanalları için çeşitli ajanların intraduktal teslim tahlil için olasılık açar. Iyi karakterize edilmiş fare modellerinde lokalize olarak verilmesi gelişmeler invazif olmayan uygulama hızlandırmak olmalıdır, insanlarda tedavi edici stratejiler hedef almıştır.

Fare meme bezi meme merkezi bir kanal içine yakınsayan kompleks duktal-alveolar ağaç oluşmaktadır. Farelerde hem de insanlarda, göğüs tümörlerinin çoğunluğu süt kanallarını astar epitel hücrelerinde kökenlidir. Şu an mevcut tedaviler intravenöz kemoterapi, radyasyon ve cerrahi içerir. Lokalize intraduktal tedaviler 1-3 araştırdı ve düşük toksisite ve yan etkiler açısından önemli avantajlar sunuyoruz edilmiştir. Bu daha yaygın olarak uygulanabilir önce Ancak, çeşitli zorluklar kalır. Fare meme bezinde benzer işlemleri gerçekleştirmek için yeteneği intraduktal müdahalelerin karakterizasyonu kolaylaştırır. Burada doğrudan fare meme kanal içine reaktiflerin teslimat için non-invaziv bir yöntem mevcut. Meme kanseri hastalarında ve fare modellerinde hem de kemoterapötik maddelerin intraduktal dağıtım daha önce hiçbir kanıt ile son derece etkili olduğu gösterilmiştirsistemik toksisite ya da uzun vadeli histopatolojik 3-6 değiştirir. Bundan başka, tümör hücre enjeksiyonu ve onkogenlerin lentiviral vector teslim önce intraduktal enjeksiyon 7-10 ile uygun olduğu gösterilmiştir. İlaç vermek için ek olarak, bu prosedür, siRNA intraduktal enjeksiyon yoluyla meme bezi genlerin yerel susturulması için yararlıdır. SiRNA çözüm küçük hacimli enjeksiyon meme bezinin anatomisi ve fizyolojisini değiştirmeden etkilidir. Laboratuarımızda yakın zamanda kendiliğinden (Brock ve diğ., Yayınlanmamış), tümör ilerlemesinin önlenmesi ile sonuçlanan meme tümörleri geliştirmektedir transgenik farelerde önemli aracı genlerin başarılı susturulması göstermiştir. Biz hiçbir görünür doku hasarı veya toksisite ile 2 haftalık aralıklarla 7 kata kadar aynı meme içine iki haftada enjeksiyonları tekrarlamak mümkün olmuştur. İntraduktal enjeksiyonları devel adresine bir yetişkin fare zamanlı olabilirgebelik, emzirme ve involüsyon sırasında ya da tümör gelişimi ve ilerlemesi sırasında sonraki aşamalarını hedef meme bezi gelişimi ile ilgili opmentally özgü olaylar. Yaşı 8 hafta gibi genç bakire kadın Laboratuvarımızda non-invaziv enjekte edilmiştir.

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

intraductal injection for localized drug delivery to the mouse mammary gland

Bu yazıda intraduktal enjeksiyon yoluyla fare meme bezinin çeşitli reaktifler teslim etmek için bir protokol açıklar. Lokalize ilaç verme ve knock-down meme epitel içinde genlerin nedeniyle bu tür gebelik ve emzirme gibi gelişim aşamaları bağımsız uygun hedefleme molekülünün eksikliği elde etmek zor olmuştur. Burada, farelerin meme intraduktal içine enjeksiyon ile erişkin herhangi bir aşamasında meme bezine reaktiflerin lokalize olarak verilmesi için kullanılan bir tekniği açıklar. Enjeksiyonlar anestezi altında, canlı farelerde gerçekleştirilen ve meme bezine bir non-invazif ve lokalize ilaç verilmesi için izin edilebilir. Ayrıca, enjeksiyon meme zarar vermeden birkaç ay boyunca tekrarlanabilir. Böyle Evans Blue gibi hayati boyalar tekniği öğrenmek için çok faydalıdır. Mavi boya intraduktal enjeksiyonu üzerine, bütün duktal ağaç gözle görünür hale gelir. Ayrıca, flüoresan etiketli reaktif maddeler de izin verirmeme bezinde görselleştirme ve dağıtım. Bu teknik, siRNA, kemoterapötik maddeler ve küçük moleküller de dahil olmak üzere çeşitli bileşikler için uyarlanabilir.

Meme kolayca saç onu (Şekil 1A) çevreleyen alanda çıkarıldıktan sonra Stereomikroskopta lokalize edilebilir. Enjeksiyon tekniği ana, o Evans mavi boya enjekte ve bezin bütünlüğünü (Şekil 1B ve C) izlemek için tavsiye edilir. Bu yaklaşım, aynı zamanda bir görsel boya tüm duktal sistemi (Şekil 1 C ve 2A) ulaşır olup olmadığını değerlendirmek için her bir bezi içine enjekte edilecek uygun hacimleri belirlenmesine izin verir. Enjekte Evans mavi görüntü bir görüntü daha sonra tüm duktal ağaç Carmine alum boya (Şekil 2B) ile boyanmış olduğu bütün monte edilmiş meme bezi, karşılaştırılabilir. Bu enjeksiyon yöntemi sağlamlığı operatör derece bağımlı olduğunu unutmayınız. Örneğin, kanal perfore bir meme yağ pedi enjeksiyonu neden olur ve çok hızlı bir çözüm enjekte duktal epitelyum cel zarar verebilirls ve enflamatuar tepkiye yol açar. Başarılı intraduktal enjeksiyon meme bezi ile lokal ilaç verilmesini sağlayan ve çoğu zaman, aksi görülen spesifik olmayan yan etkilerini azaltır. Floresan enjekte bir bezinin bir örneği Şekil 3 &#39;de resmi gösterilen siklofilin (esas teşkil etmeyen bir gen) siRNA hedef etiketlendi. <img alt="Şekil 1" src="/files/ftp_upload/50692/50692fig1.jpg" /> Şekil 1. Bir inguinal meme bezi Evans mavi boya ile meme yoluyla enjekte. 20 ul Evans mavi boya ile enjeksiyondan sonra enjeksiyon ve B öncesi meme A) Görünüm). Resim şişme ya da doku hasarı cildi açıldığında. ° C) görülmektedir, boya tüm meme duktal ağacının görselleştirme izin verir. <img alt="Şekil 2," src="/files/ftp_upload/50692/50692fig2.jpg" /> Şekil 2. Evans mavi-in analizi Tüm montajtâbi bezi. A) hayvana kesilip çıkarılır ve hemen sonra yağ kırmızı bir boya ile aynı bezinin enjeksiyonu. B) Tüm montaj boyandıktan sonra bir cam slayt üzerine yayılmış kesilmiş bezinin Örnek görüntü temizlenmişti. A) ile karşılaştırılması, enjekte edilen çözelti, meme duktal ağaç doldurur ve bez anatomik sağlam olduğunu doğrular. <img alt="Şekil 3," src="/files/ftp_upload/50692/50692fig3.jpg" /> Şekil 3,. Taze doku örneği floresan siRNA&#39;nın Intradüktal teslim. Temsili görüntü, 48 saat enjeksiyondan çıkarıldı.

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

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Fare meme bezi sulu reaktiflerin non-invazif intraduktal teslimat için bir protokol tarif edilmektedir. Bu yöntem, özellikle meme kanalları hedef meme bezlerinin meme lokalize enjeksiyon yararlanır. Bu teknik, siRNA, kemoterapötik maddeler ve küçük moleküller de dahil olmak üzere çeşitli bileşikler için uyarlanabilir.

Fare meme bezi Lokalize İlaç Teslim Intradüktal Enjeksiyon

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

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

Research

JoVE Journal

Biology

34.6K Görüntüleme.  Boston Children's Hospital and Harvard Medical School.  Fare meme bezi sulu reaktiflerin non-invazif intraduktal teslimat için bir protokol tarif edilmektedir. Bu yöntem, özellikle meme kanalları hedef meme bezlerinin meme lokalize enjeksiyon yararlanır. Bu teknik, siRNA, kemoterapötik maddeler ve küçük moleküller de dahil olmak üzere çeşitli bileşikler için uyarlanabilir. 

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

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

Fare Meme Epitel Hücreleri lactogenic Farklılaşma sırasında Mammospheres formu

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

Biyoloji

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.

Sıçanlarda Meme Bezi Morfolojisi ve Meme Kanseri Riski değişiklikler

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland ...

Evaluation of Mammary Gland Development and Function in Mouse Models

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

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

Fare Modeller Meme Bezi Geliştirme ve Fonksiyon Değerlendirme

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

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

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

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

Kemirgen Epididimal Spermatozoa&#39;lar phosphopeptide Analizi

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

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

Fare Meme Bezinin Dondurulmuş Bölümler Dolaylı İmmünofloresan

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

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

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

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

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

Fare İç Kulak Hedefli İlaç Dağıtım Yöntemleri Karşılaştırmalı İncelemesi: Bullostomy<em&gt; Karşı</em&gt; Transtimpanik Enjeksiyon

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

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

Sağlam, Bütün fare meme dokuları yalıtım hücre dışı matriks ifade ve bezi Morfoloji analizi için

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...

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

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

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

Kısmi safra kanalı ligasyonu fare: kontrollü manken yerelleştirilmiş obstrüktif Histiositoz olgusu

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

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

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

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

Trans-iç hücre embriyonik kök hücrelerin kitle enjeksiyon daha yüksek Chimerism oranları için yol açar.

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

Bovine Mammary Gland Biopsy Techniques

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

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