Name: in vivo gene delivery into mouse mammary epithelial cells through mammary intraductal injection
Uploaded: 2023-02-10T13:00:00
Description: Watch this Scientific Journal Video about In Vivo Gene Delivery into Mouse Mammary Epithelial Cells Through Mammary Intraductal Injection at JoVE.com

我们感谢Gary Chamness博士对这份手稿的有益评论。这项工作得到了国防部（DOD）CDMRP BC191649（YL）和BC191646（YL）以及美国国立卫生研究院（NIH）CA271498（YL）的支持。作者要感谢由SPORE P50CA186784支持的乳腺中心病理学核心设施，以及由CPRIT-RP180672，NIH CA125123和RR024574支持的细胞术和细胞分选核心在Joel M. Sederstrom的协助下。

所有使用小鼠的程序均按照机构动物护理和使用委员会批准的动物方案进行。在本研究中，使用9-12周龄的FVB / N或MMTV-tva 雌性小鼠。小鼠是商业或自制的（见 材料表）。使用Lenti-EGFP （FUCGW）和RCAS-Erbb2（Neu） 病毒。按照先前发表的报告10，12进行病毒制备和滴度测定。
1. 注射器准备
<ol>...

<ol>
	<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. Eds Margot. , 259-270 (2000).</li><li>Gordon, J. W., Ruddle, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+and+stable+germ+line+transmission+of+genes+injected+into+mouse+pronuclei.">Integration and stable germ line transmission of genes injected into mouse pronuclei.</a> Science. 214 (4526), 1244-1246 (1981).</li><li>Costantini, F., Lacy, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+a+rabbit+beta-globin+gene+into+the+mouse+germ+line.">Introduction of a rabbit beta-globin gene into the mouse germ line.</a> Nature. 294 (5836), 92-94 (1981).</li><li>Brinster, R. L., 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=Somatic+expression+of+herpes+thymidine+kinase+in+mice+following+injection+of+a+fusion+gene+into+eggs.">Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs.</a> Cell. 27 (1), 223-231 (1981).</li><li>Du, Z., 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=RCAS-TVA+in+the+mammary+gland:+an+in+vivo+oncogene+screen+and+a+high+fidelity+model+for+breast+transformation.">RCAS-TVA in the mammary gland: an in vivo oncogene screen and a high fidelity model for breast transformation.</a> Cell Cycle. 6 (7), 823-826 (2007).</li><li>Bu, W., 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=Mammary+precancerous+stem+and+non-stem+cells+evolve+into+cancers+of+distinct+subtypes.">Mammary precancerous stem and non-stem cells evolve into cancers of distinct subtypes.</a> Cancer Research. 79 (1), 61-71 (2019).</li><li>Bu, W., 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=Keratin+6a+marks+mammary+bipotential+progenitor+cells+that+can+give+rise+to+a+unique+tumor+model+resembling+human+normal-like+breast+cancer.">Keratin 6a marks mammary bipotential progenitor cells that can give rise to a unique tumor model resembling human normal-like breast cancer.</a> Oncogene. 30 (43), 4399-4409 (2011).</li><li>Haricharan, 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=Contribution+of+an+alveolar+cell+of+origin+to+the+high-grade+malignant+phenotype+of+pregnancy-associated+breast+cancer.">Contribution of an alveolar cell of origin to the high-grade malignant phenotype of pregnancy-associated breast cancer.</a> Oncogene. 33 (50), 5729-5739 (2014).</li><li>Bu, W., Zhang, X., Dai, H., Huang, S., 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=Mammary+cells+with+active+Wnt+signaling+resist+ErbB2-induced+tumorigenesis.">Mammary cells with active Wnt signaling resist ErbB2-induced tumorigenesis.</a> PLoS One. 8 (11), 78720 (2013).</li><li>Du, Z., 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=Introduction+of+oncogenes+into+mammary+glands+in+vivo+with+an+avian+retroviral+vector+initiates+and+promotes+carcinogenesis+in+mouse+models.">Introduction of oncogenes into mammary glands in vivo with an avian retroviral vector initiates and promotes carcinogenesis in mouse models.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (46), 17396-17401 (2006).</li><li>Siwko, S. K., 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=Lentivirus-mediated+oncogene+introduction+into+mammary+cells+in+vivo+induces+tumors.">Lentivirus-mediated oncogene introduction into mammary cells in vivo induces tumors.</a> Neoplasia. 10 (7), 653-662 (2008).</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> Journal of Mammary Gland Biology and Neoplasia. 14 (4), 401-404 (2009).</li><li>Russell, T. D., 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=Transduction+of+the+mammary+epithelium+with+adenovirus+vectors+in+vivo.">Transduction of the mammary epithelium with adenovirus vectors in vivo.</a> Journal of Virology. 77 (10), 5801-5809 (2003).</li><li>Wagner, S., Thresher, R., Bland, R., Laible, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated-virus-mediated+transduction+of+the+mammary+gland+enables+sustained+production+of+recombinant+proteins+in+milk.">Adeno-associated-virus-mediated transduction of the mammary gland enables sustained production of recombinant proteins in milk.</a> Scientific Reports. 5, 15115 (2015).</li><li>Naldini, L., 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=In+vivo+gene+delivery+and+stable+transduction+of+nondividing+cells+by+a+lentiviral+vector.">In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.</a> Science. 272 (5259), 263-267 (1996).</li><li>Coffin, J. M., Hughes, S. H., Varmus, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Retroviruses. , (1997).</li><li>Mitani, K., Kubo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+as+an+integrating+vector.">Adenovirus as an integrating vector.</a> Current Gene Therapy. 2 (2), 135-144 (2002).</li><li>McCarty, D. M., Young, S. M., Samulski, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+adeno-associated+virus+(AAV)+and+recombinant+AAV+vectors.">Integration of adeno-associated virus (AAV) and recombinant AAV vectors.</a> Annual Review of Genetics. 38, 819-845 (2004).</li><li>Bu, W., 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=Intraductal+injection+of+lentivirus+vectors+for+stably+introducing+genes+into+rat+mammary+epithelial+cells+in+vivo.">Intraductal injection of lentivirus vectors for stably introducing genes into rat mammary epithelial cells in vivo.</a> Journal of Mammary Gland Biology and Neoplasia. 25 (4), 389-396 (2020).</li><li>Dong, J., 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=Genetic+manipulation+of+individual+somatic+mammary+cells+in+vivo+reveals+a+master+role+of+STAT5a+in+inducing+alveolar+fate+commitment+and+lactogenesis+even+in+the+absence+of+ovarian+hormones.">Genetic manipulation of individual somatic mammary cells in vivo reveals a master role of STAT5a in inducing alveolar fate commitment and lactogenesis even in the absence of ovarian hormones.</a> Developmental Biology. 346 (2), 196-203 (2010).</li><li>Haricharan, 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=Mechanism+and+preclinical+prevention+of+increased+breast+cancer+risk+caused+by+pregnancy.">Mechanism and preclinical prevention of increased breast cancer risk caused by pregnancy.</a> eLife. 2, 00996 (2013).</li><li>Reddy, J. P., 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=Defining+the+ATM-mediated+barrier+to+tumorigenesis+in+somatic+mammary+cells+following+ErbB2+activation.">Defining the ATM-mediated barrier to tumorigenesis in somatic mammary cells following ErbB2 activation.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (8), 3728-3733 (2010).</li><li>Dong, J., 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=The+PR+status+of+the+originating+cell+of+ER/PR-negative+mouse+mammary+tumors.">The PR status of the originating cell of ER/PR-negative mouse mammary tumors.</a> Oncogene. 35 (31), 4149-4154 (2016).</li><li>Young, A., 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=Targeting+the+pro-survival+protein+BCL-2+to+prevent+breast+cancer.">Targeting the pro-survival protein BCL-2 to prevent breast cancer.</a> Cancer Prevention Research. 15 (1), 3-10 (2022).</li><li>Schlimgen, R., 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=Risks+associated+with+lentiviral+vector+exposures+and+prevention+strategies.">Risks associated with lentiviral vector exposures and prevention strategies.</a> Journal of Occupational and Environmental Medicine. 58 (12), 1159-1166 (2016).</li><li>Holloway, K. R., 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=Krt6a-positive+mammary+epithelial+progenitors+are+not+at+increased+vulnerability+to+tumorigenesis+initiated+by+ErbB2.">Krt6a-positive mammary epithelial progenitors are not at increased vulnerability to tumorigenesis initiated by ErbB2.</a> PLoS One. 10 (1), 0117239 (2015).</li><li>Holloway, K. R., 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=Targeting+oncogenes+into+a+defined+subset+of+mammary+cells+demonstrates+that+the+initiating+oncogenic+mutation+defines+the+resulting+tumor+phenotype.">Targeting oncogenes into a defined subset of mammary cells demonstrates that the initiating oncogenic mutation defines the resulting tumor phenotype.</a> International Journal of Biological Sciences. 12 (4), 381-388 (2016).</li><li>Hein, S. M., 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=Luminal+epithelial+cells+within+the+mammary+gland+can+produce+basal+cells+upon+oncogenic+stress.">Luminal epithelial cells within the mammary gland can produce basal cells upon oncogenic stress.</a> Oncogene. 35 (11), 1461-1467 (2015).</li><li>Annunziato, 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=In+situ+CRISPR-Cas9+base+editing+for+the+development+of+genetically+engineered+mouse+models+of+breast+cancer.">In situ CRISPR-Cas9 base editing for the development of genetically engineered mouse models of breast cancer.</a> EMBO Journal. 39 (5), 102169 (2020).</li><li>Annunziato, 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=Modeling+invasive+lobular+breast+carcinoma+by+CRISPR/Cas9-mediated+somatic+genome+editing+of+the+mammary+gland.">Modeling invasive lobular breast carcinoma by CRISPR/Cas9-mediated somatic genome editing of the mammary gland.</a> Genes &amp; Development. 30 (12), 1470-1480 (2016).</li></ol>

本文展示了将基因引入小鼠乳腺上皮细胞以模拟散发性乳腺癌的病毒导管内注射技术。通常，注射至少5周或更大的小鼠，以便在乳腺发育后开始致癌过程。此外，5周龄以下小鼠的开口通常太小而无法注射。另一方面，非常老的小鼠的有时会退化，横断可能无法揭示导管开口。同样重要的是要注意，由于种系基因突变引起的异常乳腺发育，一些转基因或敲除肿瘤模型的也可能难以注射。
<p class=...

乳腺的上皮细胞在这些腺体的功能中起着重要作用，是乳腺癌的主要起源细胞。乳腺生物学和肿瘤发生的研究经常需要将感兴趣的基因递送到这些细胞中。每个小鼠乳腺包括一棵由上皮细胞衬里的导管树，顶端有一个开口。这种结构使乳腺上皮细胞易于被病毒载体接近，病毒载体可以通过导管内注射传递到导管树的腔内1。
乳腺导管内注射技术最初用于更大的动物，如山羊、兔子和大鼠1。对于像小鼠这样小得多的动物，导管内注射需要许多精密的工具和操作人员的更多实践。小鼠导管内注射有两种方法。一种是注射1。另一种是在手术暴露后直接注射#3或#4乳腺的初级导管1。由于第一种是非侵入性的，一旦操作员经过良好的培训，就会更快，因此这种技术更常用，本文将详细描述。
与广泛使用的传统转基因小鼠模型相比，在受精卵阶段通过显微注射2，3，4引入目的基因，通过导管内病毒注射方法传递基因具有许多优点，包括：（1）避免了为每个目的基因制作转基因小鼠系的耗时过程;（2）避免目的基因对乳腺正常发育的潜在损害;（3）它在出生后的任何期望时间引入目的基因;（4）它可以很容易地共同引入一个以上的目的基因;（5）它更好地模仿了自然致瘤过程，因为感染的细胞和携带癌基因的细胞被正常细胞包围;（6）结合TVA（肿瘤病毒A，一种禽细胞表面蛋白和逆转录病毒RCAS载体的受体）技术5，可以将目的基因引入特定的细胞群中，以研究肿瘤发生的细胞起源并在乳腺6，7，8中进行细胞谱系追踪测定，9.
任何来源于逆转录病毒 10、慢病毒 11、12、腺病毒 13 和腺病毒相关病毒 （AAV）14 的载体都可用于导管内递送遗传物质。逆转录病毒和慢病毒载体永久整合到宿主基因组中;因此，它们将感兴趣的基因稳定地引入乳腺上皮细胞。虽然慢病毒可以整合到它遇到的任何细胞的基因组中15，但逆转录病毒的有效基因组整合需要靶细胞的增殖16。腺病毒和AAV载体不整合到感染细胞的基因组中，因此仅瞬时表达目的基因17，18。当目的基因只需要表达很短的时间（例如Cre）以删除絮状肿瘤抑制基因时，此功能可能是一个优势。
慢病毒、腺病毒和AAV感染它们遇到的任何小鼠细胞。但是，由于管腔上皮在很大程度上与下面的基底层绝缘，基底层通过基底膜与基质进一步分离，因此导管内注射将感染主要限制在管腔上皮细胞，这是乳腺癌起源的主要细胞。在这个管腔上皮层内，也有不同的细胞亚型，包括干细胞、祖细胞和几组分化细胞。为了感染管腔细胞群中的特定细胞亚群，可以使用TVA技术，其中禽白血病病毒衍生的RCAS载体5，10或假型慢病毒载体11选择性地感染在细胞类型特异性启动子控制下携带TVA转基因的小鼠中表达TVA的细胞，例如仅在干细胞6或某些祖细胞6中活跃的启动子6，肺泡细胞7或Wnt通路活性细胞8或Wnt通路活性细胞9。
该协议介绍了通过导管内注射病毒载体将感兴趣的基因引入乳腺上皮细胞的技术。然后证明引入基因的表达以及由此产生的增生病变和肿瘤的检测。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-HA antibody</td>
			<td>Covance</td>
			<td>MMS-101P</td>
			<td>Dilution: 1 : 1000</td>
		</tr>
		<tr>
			<td>Artificial Tears</td>
			<td>Covetrus</td>
			<td>NDC 11695-0832-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Sigma</td>
			<td>B5525</td>
			<td>microwave radiation for 45 seconds at power high of 1250W microwave oven</td>
		</tr>
		<tr>
			<td>FACSCantoII</td>
			<td>BD Biosciences</td>
			<td>V96100899</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent stereomicroscope</td>
			<td>Leica</td>
			<td>MZ16 FA</td>
			<td></td>
		</tr>
		<tr>
			<td>FUCGW lenti-virus</td>
			<td>Self-made</td>
			<td>N/A</td>
			<td>See reference # 12</td>
		</tr>
		<tr>
			<td>FVB/N</td>
			<td>The Jackson Laboratory</td>
			<td>JAX:001800</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton needle</td>
			<td>Hamilton</td>
			<td>91033</td>
			<td>autoclaved</td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Hamilton</td>
			<td>201000</td>
			<td>autoclaved</td>
		</tr>
		<tr>
			<td>LED magnifying lamp</td>
			<td>Intertek</td>
			<td>3165273</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro dissection spring scissor</td>
			<td>Roboz</td>
			<td>RS-5621</td>
			<td>autoclaved</td>
		</tr>
		<tr>
			<td>MMTV-tva</td>
			<td>Self-made</td>
			<td></td>
			<td>See reference # 10</td>
		</tr>
		<tr>
			<td>RCAS-Neu (HA)</td>
			<td>Self-made</td>
			<td>N/A</td>
			<td>See reference # 10</td>
		</tr>
		<tr>
			<td>Rodent Comboanesthetic III</td>
			<td>Veterinary Pharmacy</td>
			<td>Veterinary prescription</td>
			<td>37.6 mg/mL ketamine, 1.92 mg/mL xylazine, and 0.38 mg/mL acepromazine</td>
		</tr>
	</tbody>
</table>

in vivo gene delivery into mouse mammary epithelial cells through mammary intraductal injection

小鼠乳腺由导管树组成，导管树由上皮细胞衬里，每个的尖端都有一个开口。上皮细胞在乳腺功能中起主要作用，是大多数乳腺肿瘤的起源。将感兴趣的基因引入小鼠乳腺上皮细胞是评估上皮细胞基因功能和生成小鼠乳腺肿瘤模型的关键步骤。该目标可以通过在导管内注射携带目标基因的病毒载体到小鼠乳腺导管树中来实现。注射的病毒随后感染乳腺上皮细胞，带来感兴趣的基因。病毒载体可以是慢病毒、逆转录病毒、腺病毒或腺病毒相关病毒 （AAV）。这项研究证明了目的基因如何通过小鼠乳腺导管内注射病毒载体递送到乳腺上皮细胞中。携带 GFP 的慢病毒用于显示递送基因的稳定表达，携带 Erbb2 （HER2 / Neu）的逆转录病毒用于显示癌基因诱导的非典型增生病变和乳腺肿瘤。

这里提供的代表性数据是为了证明成功的导管内注射、成功的病毒感染以及递送的基因对乳腺肿瘤发生的影响。注射的病毒量必须根据每个实验的目的进行调整。为了说明乳腺导管树被感染的程度，需要使用大量可以成像的携带病毒的基因，例如GFP。另一方面，为了模仿自然自发的肿瘤发生，必须使用少量携带癌基因的病毒，以便只有少数细胞被感染，并在原本完全正常的乳腺领域进化为癌前病?...

Watch this Scientific Journal Video about In Vivo Gene Delivery into Mouse Mammary Epithelial Cells Through Mammary Intraductal Injection at JoVE.com

In Vivo Gene Delivery into Mouse Mammary Epithelial Cells Through Mammary Intraductal Injection

本协议描述了通过 在 导管内注射病毒载体以将感兴趣的基因递送到乳腺上皮细胞中。

体内 通过乳腺导管内注射将基因传递到小鼠乳腺上皮细胞

Mouse mammary glands comprise ductal trees, which are lined by epithelial cells and have one opening at the tip of each nipple. The epithelial cells play ...

该方法以特殊、临时和定量可控的方式将遗传物质递送到乳腺上皮细胞中。由于将遗传物质递送到乳腺上皮细胞中是特殊的，暂时和定量控制，通过这种方法生成的乳腺模型更好地模拟了天然肿瘤发生。通过将 33 号金属轮毂针切割成约 1 厘米长并将它们储存在酒精中来开始注射器制备。从酒精中取出注射器和针头，并将剩余的酒精从注射器和针头中推出。然后拆卸注射器进行空气干燥。干燥后，组装注射器。从零下80摄氏度的冰箱中取出病毒储备管，并在冰上解冻。要添加溴酚蓝，请将移液器吸头浸入溴酚蓝粉末中一厘米的深度。然后将附着的微量溴酚蓝带入病毒悬浮液中。将溴酚蓝与病毒溶液混合，上下移液10次，然后将病毒放在冰上。通过捏麻醉雌性小鼠的脚趾来检查麻醉深度。在眼睛上涂抹软膏以防止动物在麻醉下干燥。将鼠标仰卧放在温暖的垫子上，并使用胶带将所有四肢连接到工作台上。识别要注射的，并通过用剪刀修剪周围的头发来暴露它。将 70% 酒精和 iota 四根棉签交替涂抹在区域，以清洁并暴露。使用一对无菌的微解剖弹簧剪刀横断的远端尖端，直到在放大镜灯下可以看到一个小的中央导管开口。然后将10微升病毒溴酚蓝混合物装入注射器中，并在放大镜灯的帮助下小心地将针头插入开口。将整个10微升病毒注入导管树中，并将鼠标放在设置为45摄氏度的载玻片加热器上，直到鼠标完全醒来。注射病毒后三到五天打开胸腔。用剪刀沿着腹侧中线和上下肢切割皮肤。提起皮肤，露出乳腺。使用镊子和剪刀从皮肤和未注射的腺体中取出注射的乳腺作为对照。完成后，将乳腺放在载玻片上并将腺体展开至其原始形状，然后在荧光体视显微镜下观察和成像。通过暴露乳腺和观察蓝色导管树证实了导管内注射的成功。在注射慢蛋白-EGFPFUCGW病毒2至5天后，通过在荧光体视显微镜下观察整个支架制备来评估乳腺上皮细胞感染。对于病毒产生的荧光蛋白或细胞表面标志物的流式细胞术分析，收集未感染的腺体和感染的腺体并将其处理成单细胞悬液以估计病毒感染率。活化的ERBBB2在几周内导致癌前病变，在几个月内导致肿瘤。由于个体小鼠的质量，年龄，体重和力量的差异，的大小可能会有很大差异。正确修剪尖端以露出乳腺导管的开口和安装针头是成功注射病毒的重要步骤。

Research

JoVE Journal

Cancer Research

In Vivo Gene Delivery into Mouse Mammary Epithelial Cells Through Mammary Intraductal Injection

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

临床前小鼠模型的外科手术和方法<em&gt; De Novo</em&gt;乳腺癌转移

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study ...

科研

癌症研究

Sectioning Mammary Gland Whole Mounts for Lesion Identification

切片乳腺全身为病变鉴别

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and ...

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

乳腺癌细胞原位注射到小鼠乳房脂肪板

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland

通过将表达的腺病毒注射到小鼠乳腺腺中,模拟乳腺癌

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are ...

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

M1巨噬细胞-4T1小鼠乳腺癌细胞在共培养中噬菌体活性的时移2D成像

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer ...

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad

大鼠乳腺内皮细胞移植到间膜白脂肪垫

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

人类乳腺上皮细胞致癌转化的体外评价

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能 体外 模型

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

一种用于乳腺肿瘤微环境活体内成像的无标记分割方法

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

Intraductal Delivery and X-ray Visualization of Ethanol-Based Ablative Solution for Prevention and Local Treatment of Breast Cancer in Mouse Models

导管内递送和X射线可视化乙醇烧蚀溶液在小鼠模型中预防和局部治疗乳腺癌

Breast cancer is the most prevalent cancer and the second-leading cause of cancer-related death for women in the USA. For high-risk women, prophylactic ...

体内 通过乳腺导管内注射将基因传递到小鼠乳腺上皮细胞

体内通过乳腺导管内注射将基因传递到小鼠乳腺上皮细胞