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The present protocol describes intraductal injection of viral vectors via the teat to deliver genes of interest into the mammary epithelial cells.
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.
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....
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,<.......
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.......
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.......
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.
....Name | Company | Catalog Number | Comments |
Anti-HA antibody | Covance | MMS-101P | Dilution: 1 : 1000 |
Artificial Tears | Covetrus | NDC 11695-0832-1 | |
Bromophenol blue | Sigma | B5525 | microwave radiation for 45 seconds at power high of 1250W microwave oven |
FACSCantoII | BD Biosciences | V96100899 | |
Fluorescent stereomicroscope | Leica | MZ16 FA | |
FUCGW lenti-virus | Self-made | N/A | See reference # 12 |
FVB/N | The Jackson Laboratory | JAX:001800 | |
Hamilton needle | Hamilton | 91033 | autoclaved |
Hamilton syringe | Hamilton | 201000 | autoclaved |
LED magnifying lamp | Intertek | 3165273 | |
Micro dissection spring scissor | Roboz | RS-5621 | autoclaved |
MMTV-tva | Self-made | See reference # 10 | |
RCAS-Neu (HA) | Self-made | N/A | See reference # 10 |
Rodent Comboanesthetic III | Veterinary Pharmacy | Veterinary prescription | 37.6 mg/mL ketamine, 1.92 mg/mL xylazine, and 0.38 mg/mL acepromazine |
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