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.
