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

We thank Dr. Gary Chamness for his helpful comments on this manuscript. This work was supported by the Department of Defense (DOD) CDMRP BC191649 (YL) and BC191646 (YL) as well as the National Institutes of Health (NIH) CA271498 (YL). The authors would like to thank the Breast Center Pathology Core Facility supported by SPORE P50CA186784, and the Cytometry and Cell Sorting Core supported by CPRIT-RP180672, NIH CA125123, and RR024574 with the assistance of Joel M. Sederstrom.

All procedures using mice were performed in compliance with the Institutional Animal Care and Use Committee-approved animal protocol. For the present study, 9-12-week-old FVB/N or MMTV-tva female mice were used. The mice were obtained commercially or self-made (see Table of Materials). The Lenti-EGFP (FUCGW) and RCAS-Erbb2 (Neu) viruses were used. Virus preparation and titer determination were performed following the previously published reports10,<...

<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>

This article demonstrates the viral intraductal injection technique for introducing genes into mouse mammary epithelial cells for modeling sporadic breast cancer. Usually, mice of at least 5 weeks or older are injected so that the oncogenic process starts after the mammary gland is developed. Besides, the nipple opening of mice younger than 5 weeks old is often too small for injection. On the other hand, the nipples of very old mice are sometimes degenerated, and transection may fail to reveal a ductal opening. It is als...

Epithelial cells of mammary glands play a major role in the function of these glands and are the major cell of origin of breast cancer. Studies of mammary gland biology and tumorigenesis frequently need the delivery of gene(s) of interest into these cells. Each mouse mammary gland comprises a ductal tree lined by epithelial cells with a single opening at the tip of the nipple. This structure makes the mammary epithelial cells easily accessible to viral vectors, which can be delivered into the lumen of a ductal tree via intraductal injection1.
The technique of mammary intraductal injection was originally used for much larger animals such as goats, rabbits, and rats1. For a much smaller animal such as mice, intraductal injection needs many delicate tools and more practices of the operators. There are two approaches for mouse intraductal injection. One is up-the-teat injection1. Another one is the direct injection of the primary duct of #3 or #4 mammary gland after surgical exposure1. Since the first one is non-invasive and faster once the operator has been well-trained, this technique is more commonly used and will be described in detail in this article.
Compared to the widely used traditional transgenic mouse models, in which the gene of interest is introduced at the stage of fertilized eggs through microinjection2,3,4, gene delivery through the intraductal virus injection method has many advantages, including: (1) it avoids the time-consuming process of making a transgenic mouse line for each gene of interest; (2) it avoids potential impairment on the normal development of mammary glands imposed by the gene of interest; (3) it introduces the gene of interest at any desired time after birth; (4) it can easily co-introduce more than one gene of interest; (5) it better mimics the natural tumorigenic process because the infected and thus oncogene-carrying cells are surrounded by normal cells; and (6) in combination with the TVA (tumor virus A, an avian cell surface protein and the receptor for retrovirus RCAS vector) technology5, the gene of interest can be introduced into a specific cell population to study the cell origin of tumorigenesis and to conduct cell lineage-tracing assays in the mammary glands6,7,8,9.
Any vectors derived from retrovirus10, lentivirus11,12, adenovirus13, and adenovirus-associated virus (AAV)14 may be used for intraductal delivery of genetic materials. Retrovirus and lentivirus vectors integrate into the host genome permanently; thus, they introduce genes of interest stably into mammary epithelial cells. While lentivirus can integrate into the genome of any cell it encounters15, the efficient genomic integration of retrovirus needs the proliferation of the target cells16. The adenoviral and AAV vectors do not integrate into the genome of infected cells and, therefore, only transiently express the gene of interest17,18. This feature can be an advantage when the gene of interest needs to be expressed for only a short amount of time, such as Cre, for deleting a floxed tumor suppressor gene.
Lentivirus, adenovirus, and AAV infect any mouse cells they encounter. But since luminal epithelium is largely insulated from the underlying basal layer, which is further separated from the stroma by the basement membrane, intraductal injection limits the infection largely to luminal epithelial cells, the primary cell of origin of breast cancer. Within this luminal epithelial layer, there are also distinct cell subtypes, including stem cells, progenitor cells, and several groups of differentiated cells. To infect specific cell subsets within the luminal cell population, the TVA technology may be used, with which avian leukosis virus-derived RCAS vectors5,10 or pseudotyped lentiviral vectors11 selectively infect the cells that express TVA in mice that carry a tva transgene under the control of a cell type-specific promoter, such as a promoter that is active only in stem cells6 or certain progenitors6,7 or alveolar cells8 or Wnt-pathway active cells9.
This protocol presents the technique of introducing genes of interest into mammary epithelial cells through intraductal injection of a viral vector. The detection of expression of the introduced genes and the resulting hyperplastic lesions and tumors are then demonstrated.

<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

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 a major role in mammary gland function and are the origin of most mammary tumors. Introducing genes of interest into mouse mammary epithelial cells is a critical step in evaluating gene function in epithelial cells and generating mouse mammary tumor models. This goal can be accomplished through the intraductal injection of a viral vector carrying the genes of interest into the mouse mammary ductal tree. The injected virus subsequently infects mammary epithelial cells, bringing in the genes of interest. The viral vector can be lentiviral, retroviral, adenoviral, or adenovirus-associated viral (AAV). This study demonstrates how a gene of interest is delivered into mammary epithelial cells through mouse mammary intraductal injection of a viral vector. A lentivirus carrying GFP is used to show stable expression of a delivered gene, and a retrovirus carrying Erbb2 (HER2/Neu) is used to demonstrate oncogene-induced atypical hyperplastic lesions and mammary tumors.

Representative data are presented here to demonstrate successful intraductal injection, successful viral infection, and the impact of the delivered genes on mammary tumorigenesis. The amount of virus injected must be tailored to the purpose of each experiment. To illustrate how extensively the mammary duct tree can be infected, a large amount of virus carrying genes that can be imaged, such as GFP, needs to be used. On the other hand, to mimic the natural spontaneous tumorigenesis, a small amount of virus carrying an onc...

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

The present protocol describes intraductal injection of viral vectors via the teat to deliver genes of interest into the mammary epithelial cells.

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

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 ...

This method delivers the genetic material into the mammary gland epithelial cells in a special, temporary and quantitatively controllable manner. Since the delivery of genetic material into the mammary epithelial cells is special, temporarily and quantitatively controlled, mammary models generated by this method better mimic natural tumor genesis. Begin syringe preparation by cutting the 33 gauge metal hub needles to approximately one centimeter length and storing them in alcohol.Remove syringes and needles from the alcohol and propel the remaining alcohol out of the syringe and needles. Then disassemble the syringe for air drying. After drying, assemble the syringe.Take out the viral stock tubes from the minus 80 degrees Celsius freezer and thaw them on ice. To add bromophenol blue, dip the pipette tip to a depth of one centimeter into the bromophenol blue powder. Then bring the attached trace amount of bromophenol blue into the virus suspension.Mix the bromophenol blue with the virus solution by pipetting up and down 10 times, before placing the virus on ice. Check the depth of anesthesia by pinching the toe of an anesthetized female mouse. Apply ointment on the eyes to prevent dryness while the animal is under anesthesia.Put the mouse in a supine position on a warm pad and attach all four limbs to the bench using adhesive tape. Identify the nipple to be injected and expose it by trimming the surrounding hair using scissors. Apply 70%alcohol and iota four swabs in three alternating rounds to the nipple area to clean and expose the nipple.Transect the distal tip of a nipple using a pair of sterilized micro dissection spring scissors, until a small central ductal opening can be seen under a magnifier lamp. Then load 10 microliters of the virus bromophenol blue mixture into the syringe and carefully insert the needle into the nipple opening with the help of the magnifier lamp. Inject the entire 10 microliters of the virus into the duct tree and place the mouse on a slide warmer set at 45 degrees Celsius until the mouse fully wakes up.Open the thoracic cavity three to five days after the virus injection. Cut the skin along the ventral middle line and the upper and lower limbs using scissors. Lift the skin and expose the mammary glands.Remove the injected mammary gland from the skin and an uninjected gland as a control using forceps and scissors. Once done, place the mammary gland on a glass slide and spread the gland to its original shape before observing and imaging it under a fluorescent stereo microscope. The success of intraductal injection was confirmed by exposing the mammary gland and observing a blue ductal tree.After two to five days of the lenti-EGFPFUCGW virus injection, the mammary epithelial cell infection was assessed by observing whole mount preparation under a fluorescent stereo microscope. For flow cytometry analysis of the fluorescent proteins or cell surface markers produced by the virus the non-infected glands and infected glands were collected and processed into a single cell suspension to estimate the rate of viral infection. Activated ERBBB2 led to pre-cancerous lesions in a few weeks and tumors in a few months.The size of the nipples can vary significantly due to the differences in mass, age, body weight and strength among individual mice. Properly trimming the tip of the nipple to expose the opening of the mammary duct and the fitting the needle is an important step for successfully injecting the virus.

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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

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

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

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

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

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 ...