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

Summary

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

Abstract

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.

Introduction

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....

Protocol

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 reports^10,<.......

Discussion

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.......

Materials

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