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 reports10,12.
1. Syringe preparation
<ol>
	<li>Cut the 33 G metal hub needles (see Table of Materials) to approximately 1 cm in length. Store the needles and the 50 &#181;L syringe in 70% alcohol after autoclaving.</li>
	<li>Take out syringes and needles from the alcohol. Propel the remaining alcohol out of the syringe and needle. Disassemble the syringe for air drying on an autoclaved absorbent bench pad.</li>
	<li>Assemble the syringe after drying.</li>
</ol>
2. Virus preparation
<ol>
	<li>Take out one or several viral stock tubes as needed from the -80 &#730;C freezer and thaw the virus on ice. 
	NOTE: Depending on the number of cells to be infected, the virus may need to be diluted into intended titers using 1x PBS at this time.</li>
	<li>Add bromophenol blue by dipping the pipette tip to a depth of 1 cm into the bromophenol blue powder (see Table of Materials). Then, bring the attached trace amount of bromophenol blue into the virus suspension. 
	NOTE: For the present study, the viral stock is usually 200 &#181;L per tube, so the final dye concentration is approximately 0.2% (0.2 &#181;g/100 &#181;L). Bromophenol blue should be sterilized with microwave radiation for 45 seconds at high power of 1250 W&#160;(see&#160;Table of Materials). Ensure to carry whole procedure in a sterile environment.</li>
	<li>Mix the bromophenol blue with the virus solution by pipetting up and down 10 times.</li>
	<li>Place the virus on ice and bring the ice bucket to the vivarium.</li>
</ol>
3. Animal preparation
<ol>
	<li>Anesthetize a female mouse by intraperitoneal injection of 2 &#181;L/g of anesthetic (37.6 mg/mL ketamine, 1.92 mg/mL xylazine, and 0.38 mg/mL acepromazine, see Table of Materials). Check the depth of anesthesia with a toe pinch. Apply ointment on the eyes to prevent dryness while under anesthesia. 
	NOTE: The mouse must be non-responsive to toe&#160;pinch under good anesthesia. Isoflurane may also be used to anesthetize the mice.</li>
	<li>Put the mouse in a supine position on a warm pad and attach all four limbs to the bench using adhesive tape.</li>
	<li>Identify one nipple (or more) to be injected (number 4 is preferred for the present study). Expose it by trimming the surrounding hair using a pair of scissors.</li>
	<li>Apply&#160;70% alcohol and iodophor swabs to the nipple area in three rounds to&#160;clean and expose the nipple.</li>
</ol>
4. Intraductal injection of virus
NOTE: A magnifying lamp may be used to help visualize the nipple opening.
<ol>
	<li>Transect the distal tip of a nipple using a pair of sterile&#160;microdissection spring scissors, until a small central ductal opening can be seen under a magnifier lamp (see Table of Materials).</li>
	<li>Load 10 &#181;L of virus/bromophenol blue mixture into the syringe. 
	NOTE: Lenti-EGFP (FUCGW) and RCAS-Erbb2 (Neu) are used in this demonstration.</li>
	<li>Carefully insert the needle into the nipple opening with the help of the magnifier lamp. The orientation of the needle is adjusted slightly from medial to lateral to align with the main duct.</li>
	<li>Inject the entire 10 &#181;L of the virus into the duct tree. 
	NOTE: The ducts under the skin should turn blue, if the injection is successful. Any resistance or the appearance of a localized color change indicates the failure of the injection.</li>
	<li>Put the mouse on a slide warmer set at 45 &#176;C until the mouse fully wakes up(~30-60 min).</li>
</ol>
5. Detection of infected cells by a fluorescent stereomicroscope
<ol>
	<li>Three to five days after injection of the virus carrying GFP or other fluorescent genes, euthanize the mouse by over-dosing it with 4 uL/g of anesthetic (37.6 mg/mL ketamine, 1.92 mg/mL xylazine, and 0.38 mg/mL acepromazine).</li>
	<li>Open the thoracic cavity. Cut the skin along the ventral middle line and along the upper and lower limbs using a pair of scissors. Lift the skin and expose the mammary glands.</li>
	<li>Remove the injected mammary gland from the skin using a pair of forceps and scissors. Also, remove an un-injected gland as a control.</li>
	<li>Place the mammary glands on glass slides and spread the glands to their original shape.</li>
	<li>Observe and image the glands under a fluorescent stereomicroscope.</li>
</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>

Both the authors declare no conflicts of interest.

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 also important to note that the nipples of some transgenic or knockout tumor models may also be difficult to inject due to abnormal mammary development caused by germline genetic mutations.
Besides practicing the use of standard personal protective equipment (PPE) in handling viruses, caution must be taken to avoid accidental needle stings in the experimenter&#39;s hands. While there is no evidence of avian leukosis virus infecting humans, lentiviruses used for cancer modeling in rodents can infect humans as well. This protection from accidental infection is especially important when the virus carries a potent oncogene25.
When multiple genes of interest need to be delivered into epithelial cells, they can be delivered by a mixture of different vectors carrying each individual gene or by a vector designed to carry all the genes to be tested. The former method can take advantage of previously produced viruses, but co-infection only occurs in a small subset of the infected population. On the other hand, the latter approach often requires constructing new viruses, but co-infection is guaranteed.
Compared to the genetically engineered mouse models (GEMMs), the intraductal injection technique provides better spatiotemporal flexibility to deliver the gene of interest into mammary epithelial cells. This flexibility facilitates better mimicking tumorigenesis in patients since most breast cancers originate from somatic cells that suffer genetic changes during adult stages5. Spatiotemporal control can also help to study the contribution of cell sub-types to breast tumorigenesis6,7,9,26,27,28.
The intraductal injection technique has been used mostly to deliver exogenous genes driven by artificial promoters with a viral vector. When an oncogene is delivered this way, its transcription differs from that of a naturally mutated endogenous proto-oncogene. And this difference may subsequently impact cancer formation, progression, and response to therapy. To overcome this concern, vectors carrying CRISPR/Cas9-based gene-editing components can be used to edit endogenous genes of interest29,30. We have recently improved this strategy and made it very flexible and efficient (Bu et al., unpublished results). This progress in gene editing makes the intraductal injection technique an even more powerful tool for cancer modeling.
In conclusion, intraductal injection of viral vectors is a powerful technique for generating mouse models of breast cancer that closely mimic human breast cancer formation. The combination of this technique with the TVA technology and CRISPR editing unleashes even more potential for this versatile method in understanding breast cancer formation. These models also provide valuable resources for testing new strategies in breast cancer prevention.

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><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 oncogene must be used so that only a few cells are infected and will evolve to precancerous lesions, and eventually invasive cancer, in a field of the otherwise completely normal mammary gland.
The success of intraductal injection can be immediately confirmed by exposing the mammary gland and observing a blue ductal tree (Figure 1). Two to five days after injection of the Lenti-EGFP (FUCGW) virus (~106 IUs)12, mammary epithelial cell infection can be assessed by whole mount preparation followed by observing under a fluorescent stereomicroscope (Figure 2). Alternatively, for flow cytometry analysis of the fluorescent proteins or cell surface markers produced by the virus6,7,19, the infected glands and non-infected glands (as negative controls) may be collected and processed into a single-cell suspension so that the rate of viral infection can be estimated (Figure 3). Another method to test/quantify virus infection is to fix the infected and non-infected control glands using 4% paraformaldehyde (or formalin), process them into paraffin-embedded blocks, and stain the resulting sections for the virally produced gene products and epitope tags such as HA and FLAG20,21.
Whether the virally infected cells will expand and form precancerous lesions, and then tumors, depends on the potency of the oncogene(s) delivered and the amount of virus injected. For a potent oncogene such as PyMT and activated RAS, early lesions form in a few days, and tumors appear in a few weeks10 (Bu et al., unpublished observations). Activated ERBB2 leads to precancerous lesions in a few weeks and tumors in a few months10,22,23 (Figure 4), while activated PIK3CA leads to tumors with a median latency of approximately 5 months24. On the other hand, Wnt1 causes tumors rather slowly: only about 20% of the infected mice developed tumors after 20 months21.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64718/64718fig01.jpg" /> 
Figure 1: A successfully injected mammary ductal tree. The image was captured immediately after the intraductal injection of the number 4 mammary gland of a 9-week-old FVB/N female mouse. The arrow indicates the location of the number 4 nipple. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64718/64718fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64718/64718fig02.jpg" /> 
Figure 2: Detection of infected cells by fluorescent stereomicroscopy. (A) An un-injected contralateral gland is used as a control. (B) An image captured under a fluorescent stereomicroscope shows the infected mammary duct tree. The number 3 mammary gland of a 10-week-old FVB/N mouse was intraductally injected with the Lenti-EGFP (FUCGW) virus (~106 IUs). The injected mammary gland was collected 5 days after the injection. Scale bar: 1 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64718/64718fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64718/64718fig03.jpg" /> 
Figure 3: Quantifying infected cells by flow cytometry analysis for EGFP produced by lentivirus. The phycoerythrin (PE) channel was used to reveal the auto-fluorescent signal. The contralateral uninjected mammary glands were used as the negative control. The injected mammary glands from a 12-week-old FVB/N female mouse were collected 2.5 days after intraductal injection of Lenti-EGFP (~106 IUs/gland) and processed into a single-cell suspension. The single-cell suspension was then analyzed by flow cytometry to detect the GFP-positive cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64718/64718fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64718/64718fig04.jpg" /> 
Figure 4: Immunohistochemical confirmation of virus-expressed oncogene product in an early lesion and a tumor. (A) An early lesion-containing #4 mammary gland was collected 14 days after intraductal injection of 106 IUs of RCAS-Neu (HA) into MMTV-tva mice. Immunohistochemical staining was used to detect the HA tag. (B) A tumor was collected 1 year after intraductal injection of 104 IUs of RCAS-Neu (HA) into a #4 gland of MMTV-tva mice. Mouse age at injection: 12 weeks. Scale bar: 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64718/64718fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

in-vivo-gene-delivery-into-mouse-mammary-epithelial-cells-through

Nanyang Technological University

Research

JoVE Journal

Cancer Research

1.3K Views.  Baylor College of Medicine. Deze methode levert het genetisch materiaal op een speciale, tijdelijke en kwantitatief controleerbare manier af in de borstepitheelcellen. Omdat de afgifte van genetisch materiaal in de borstepitheelcellen speciaal, tijdelijk en kwantitatief gecontroleerd is, bootsen borstmodellen die door deze methode worden gegenereerd de natuurlijke tumorgenese beter na. Begin met het bereiden van de spuit door de metalen naafnaalden van 33 gauge tot ongeveer een centimeter lang te snijden en ze in alcoho...

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

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

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs&#39; metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

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

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are instrumental in gaining insights into these complex processes. Although many mouse models have been developed to study contributions of various oncogenic events and cells of origin to breast tumorigenesis, these models are often not cell-type or organ specific or cannot induce the initiation of mammary tumorigenesis in a temporally controlled manner. Here we describe a protocol to generate a new type of breast cancer mouse models based on the intraductal injection of Cre-expressing adenovirus (Ad-Cre) into mouse mammary glands (MGs). Due to the direct injection of Ad-Cre into mammary ducts, this approach is MG specific, without any unwanted cancer induction in other organs. The intraductal injection procedure can be performed in mice at different stages of their MG development (thus, it permits temporal control of cancer induction, starting from ~3-4 weeks of age). The cell-type specificity can be achieved by using different cell-type-specific promoters to drive Cre expression in the adenoviral vector. We show that luminal and basal mammary epithelial cells (MECs) can be tightly targeted for Cre/loxP-based genetic manipulation via an intraductal injection of Ad-Cre under the control of the Keratin 8 or Keratin 5 promoter, respectively. By incorporating a conditional Cre reporter (e.g., Cre/loxP-inducible Rosa26-YFP reporter), we show that MECs targeted by Ad-Cre, and tumor cells derived from them, can be traced by following the reporter-positive cells after intraductal injection.

The goal of this protocol is to describe a new breast cancer modeling approach based on the intraductal injection of Cre-expressing adenovirus into mouse mammary glands. This approach allows both cell-type- and organ-specific manipulation of oncogenic events in a temporally controlled manner.

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

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 mastectomy is the most effective primary prevention strategy. Prophylactic mastectomy is an aggressive surgical procedure that completely removes the mammary epithelial cells from which breast cancer arises along with the surrounding tissue. We seek to develop a minimally invasive intraductal procedure as an alternative to prophylactic mastectomy to locally ablate the mammary epithelial cells before they can become malignant. We and others have developed an intraductal delivery procedure to reach and treat these epithelial cells in rodent models of breast cancer. While the mouse mammary gland with a single non-anastomosed ductal tree opening at the nipple has a much less complex and tortuous architecture than the human breast, chemically induced and genetically engineered mouse models of breast cancer are valuable to produce proof-of-concept studies of new preventative strategies. Here, we describe a procedure for intraductal delivery of an ethanol-based ablative solution containing micro-CT/X-ray tantalum-based contrast agent within the mouse mammary ductal tree for the therapeutic purpose of primary prevention of breast cancer. Intraductal delivery of aqueous reagents (e.g., cytotoxic compounds, siRNAs, AdCre) has been previously described in mouse models. Thus, we focus our protocol description on methodological modifications and unique experimental considerations for optimizing delivery of ethanol, for minimizing local and systemic side effects of ethanol administration, and for in vivo visualization of ductal tree filling via micro-CT/fluoroscopy imaging. Visualization of the ductal tree immediately after injection of a contrast-containing solution allows for confirmation of complete filling or unsuccessful outcomes such as underfilling or overfilling. This procedure can be applied for delivery and imaging of other ablative compounds aimed at either preventing tumor formation or locally treating early-stage tumors accessible via the ductal tree.

A method of intraductal injection of reagents for an ethanol-based ablative solution to the mouse mammary ductal tree for in vivo imaging and breast cancer prevention is described. Injection directly into the nipple opening allows for targeting mammary epithelial cells with minimal collateral tissue damage.

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 ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Medicine

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Intraductal Delivery to the Rabbit Mammary Gland

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and adverse effects1,2,3,4,5,6,7. However, several challenges remain before these treatments can be applied more widely. The development and validation of intraductal therapeutics in an appropriate animal model facilitate the development of intraductal therapeutic strategies for patients. While the mouse mammary gland has been widely used as a model system of mammary development and tumorigenesis, the anatomy is distinct from the human gland. A larger animal model, such as the rabbit, may serve as a better model for mammary gland structure and intraductal therapeutic development. In contrast to mice, in which ten ductal trees are spatially distributed along the body axis, each terminating in a separate teat, the rabbit mammary gland more closely resembles the human gland, with multiple overlapping ductal systems that exit through separate openings in one teat. Here, we present minimally invasive methods for the delivery of reagents directly into the rabbit mammary duct and for visualization of the delivery itself with high-resolution ultrasound imaging.

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

Bioengineering

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 ...

Biology

Longitudinal Intravital Microscopy Using a Mammary Imaging Window with Replaceable Lid

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in combination with skin flap surgery or implantation of imaging windows has been used to study the dynamics of the healthy mammary gland at different developmental stages. Most mammary imaging technologies are limited to a time frame of hours to days, whereas the majority of mammary gland remodeling processes occur in time frames of days to weeks. To study mammary gland remodeling, methods that allow optical access to the tissue of interest for extended time frames are required. Here, an improved version of the titanium mammary imaging window with a replaceable lid (R.MIW) is described that allows high-resolution imaging of the mammary gland with a cellular resolution for up to several weeks. Importantly, the R.MIW provides tissue access over the entire duration of the intravital imaging experiment and could therefore be used for local tissue manipulation, labeling, drug administration, or image-guided microdissection. Taken together, the R.MIW enables high-resolution characterization of the cellular dynamics during mammary gland development, homeostasis, and disease.

This protocol describes a novel mammary imaging window with a replaceable lid (R.MIW). Intravital microscopy after implantation of the R.MIW allows for longitudinal and multi-day imaging of the healthy and diseased mammary gland with a cellular resolution during the different developmental stages.

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in ...

Intravital Subcellular Microscopy of the Mammary Gland

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory mechanisms. During pregnancy, the gland expands from a primitive ductal tree embedded in a fat pad to a highly branched alveolar network primed for the formation and secretion of colostrum and milk. Post-partum, the gland supplies all the nutrients required for neonatal survival, including membrane-coated lipid droplets (LDs), proteins, carbohydrates, ions, and water. Various milk components, including lactose, casein micelles, and skim-milk proteins, are synthesized within the alveolar cells and secreted from vesicles by exocytosis at the apical surface. LDs are transported from sites of synthesis in the rough endoplasmic reticulum to the cell apex, coated with cellular membranes, and secreted by a unique apocrine mechanism. Other preformed constituents, including antibodies and hormones, are transported from the serosal side of the epithelium into milk by transcytosis. These processes are amenable to intravital microscopy because the mammary gland is a skin gland and, therefore, directly accessible to experimental manipulation. In this paper, a facile procedure is described to investigate the kinetics of LD secretion in situ, in real-time, in live anesthetized mice. Boron-dipyrromethene (BODIPY)665/676&#160;or monodansylpentane are used to label the neutral lipid fraction of transgenic mice, which either express soluble EGFP (enhanced green fluorescent protein) in the cytoplasm, or a membrane-targeted peptide fused to either EGFP or tdTomato. The membrane-tagged fusion proteins serve as markers of cell surfaces, and the lipid dyes resolve LDs &#8805; 0.7 &#181;m. Time-lapse images can be recorded by standard laser scanning confocal microscopy down to a depth of 15-25 &#181;m or by multiphoton microscopy for imaging deeper in the tissue. The mammary gland may be bathed with pharmacological agents or fluorescent dyes throughout the surgery, providing a platform for acute experimental manipulations as required.

The present protocol describes a facile technique for the intravital imaging of the lactating mouse mammary gland by laser scanning confocal and multiphoton microscopy.

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory ...

Intraductal Injection: Delivering Injection Mix into the Ducts of the Mouse Mammary Gland

Source: Xiang, D., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/59502/modeling-breast-cancer-via-an-intraductal-injection-cre-expressing">Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland.</a> J. Vis. Exp (2019).
This video describes an intraductal injection technique where we deliver injection mix into the ducts of mouse mammary glands.

Source: Xiang, D., et al. Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland. J. Vis. Exp ...

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video