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 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 microinjection^2,3,4, 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 glands^6,7,8,9.

Any vectors derived from retrovirus¹⁰, lentivirus^11,12, 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 interest^17,18. 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 vectors^5,10 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 progenitors^6,7 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.

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^10,12.

1. Syringe preparation

1. Cut the 33 G metal hub needles (see Table of Materials) to approximately 1 cm in length. Store the needles and the 50 µL syringe in 70% alcohol after autoclaving.
2. 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.
3. Assemble the syringe after drying.

2. Virus preparation

1. Take out one or several viral stock tubes as needed from the -80 ˚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.
2. 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 µL per tube, so the final dye concentration is approximately 0.2% (0.2 µg/100 µL). Bromophenol blue should be sterilized with microwave radiation for 45 seconds at high power of 1250 W (see Table of Materials). Ensure to carry whole procedure in a sterile environment.
3. Mix the bromophenol blue with the virus solution by pipetting up and down 10 times.
4. Place the virus on ice and bring the ice bucket to the vivarium.

3. Animal preparation

1. Anesthetize a female mouse by intraperitoneal injection of 2 µL/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 pinch under good anesthesia. Isoflurane may also be used to anesthetize the mice.
2. Put the mouse in a supine position on a warm pad and attach all four limbs to the bench using adhesive tape.
3. 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.
4. Apply 70% alcohol and iodophor swabs to the nipple area in three rounds to clean and expose the nipple.

4. Intraductal injection of virus

NOTE: A magnifying lamp may be used to help visualize the nipple opening.

1. Transect the distal tip of a nipple using a pair of sterile microdissection spring scissors, until a small central ductal opening can be seen under a magnifier lamp (see Table of Materials).
2. Load 10 µL of virus/bromophenol blue mixture into the syringe.
   NOTE: Lenti-EGFP (FUCGW) and RCAS-Erbb2 (Neu) are used in this demonstration.
3. 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.
4. Inject the entire 10 µL 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.
5. Put the mouse on a slide warmer set at 45 °C until the mouse fully wakes up(~30-60 min).

5. Detection of infected cells by a fluorescent stereomicroscope

1. 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).
2. 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.
3. Remove the injected mammary gland from the skin using a pair of forceps and scissors. Also, remove an un-injected gland as a control.
4. Place the mammary glands on glass slides and spread the glands to their original shape.
5. Observe and image the glands under a fluorescent stereomicroscope.

Results

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...

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