All animal experiments were approved by the Wistar Institute Animal Care and Use Committee.

1. Generation and Maintenance of Transgenic Mice

1. Breed LSL-K-ras^{tm4Tyj 26} and Trp53^{tm1Brn 27} (obtained from NCI mouse models of human cancer consortium on a mixed background) to a full C57BL/6 background ²⁸ by backcrossing at least 10 generations with C57BL/6 mice. To track tumor metastasis, breed B6.129X1-Gt(ROSA)26Sor^tm1(EYFP)Cos/J (LSL-EYFP, obtained from The Jackson Laboratory on a full C57BL/6 background) with double transgenic LSL-K-ras^G12D/+ p53^loxP/loxP mice.
   Note: Transgenic LSL-K-ras^G12D/+ p53^loxP/loxP mice have loxP sites flanking a transcriptionally silenced allele of oncogenic K-ras and the endogenous p53 locus, so that upon Cre-mediated excision, overexpression of an oncogenic K-ras mutant and ablation of p53 is achieved.
   Note: The LSL-EYFP mouse contains a stop codon flanking a gene for enhanced yellow fluorescent protein (YFP) that upon Cre-mediated excision results in the expression of YFP in the tissues where the YFP stop cassette is excised.
   1. Breed transgenic mice to obtain LSL-K-ras^G12D/+ p53^loxP/loxP mice or LSL-K-ras^G12D/+ p53^loxP/loxP LSL-EYFP mice for intraductal injections.
      Note: Mice are bred as homozygous for p53^loxP/loxP and heterozygous for LSL-K-ras^G12D/+ because mice with a homozygous deletion of K-ras die in utero. Use naïve virgin females at least six-weeks old for intraductal injections.
      Note: The primers for genotyping homozygous floxed p53 allele are p53–T010-fwd (5'-AAGGGGTATGAGGGACAAGG-3') and p53-T011-rev (5'-GAAGACAGAAAAGGGGAGGG-3'). They produce a wild type allele at 391 bp and the p53 floxed allele at 461 bp^29,30.
      Note: The primers to detect the mutant form of K-ras are oIMR8273 (5'-CGCAGACTGTAGAGCAGCG-3') and oIMR8274 (5'-CCATGGCTTGAGTAAGTCTGC-3'). They produce the mutant band detected at 600bp.
      Note: For the YFP reporter triple transgenic mice, the primers to detect the ROSA cassette (5'-AAGACCGCGAAGAGTTTGTC-3'), the wild type allele (5'-GGAGCGGGAGAAATGGATATG-3'), and a shared allele (5'-AAAGTCGCTCTGAGTTGTTAT-3') result in the amplification of bands at 320 bp for floxed allele and 600 bp for the wild type allele.

2. Surgical Preparation

1. Clean surgical materials with 75% EtOH and autoclave them before and after all injections.
2. Perform surgery on a clean uncluttered laboratory bench in a sanitized room within an animal facility. Wipe down all surfaces including the stage and dials of the surgical microscope with a broad-spectrum disinfectant solution followed by 75% EtOH.
3. Weigh and anesthetize mice by intraperitoneal injection of a mix of ketamine (80-100 mg/kg) and xylazine (8-10 mg/kg) in sterile saline.
4. Gently place mice back into their cages undisturbed for 5 min while they go under anesthesia. During this time generate virus precipitates (see Protocol 3).
5. Verify lack of response to pain by toe pinching. Gently cover the eyes of anesthetized mice with veterinary ointment to prevent excessive corneal drying.
6. To prevent hypothermia, place anesthetized mice onto a heating pad set to low heat during the surgical procedure and until they begin to recover.
7. For the management of pain, administer mice meloxicam subcutaneously at 1 mg/kg before the surgery and 24 hr after.

3. Generation of Virus Precipitates

CAUTION: Adenovirus vectors, although they have been modified and are unable to replicate, pose the risk of infection. Handle adenovirus with caution. All personnel should be appropriately trained according to the institution's guidelines for handling BSL2 agents After intraductal injection, dispose of adenovirus in accordance with BSL2 guidelines.

1. Store adenovirus concentrated virus stocks at -80 °C frozen in aliquots of 4 x 10⁸ pfu each, sufficient for injecting 16 animals with 3 μl of 2.5 x 10⁷ pfu of adenovirus particles.
2. Store adenovirus aliquots on dry ice until approximately 15-20 min before beginning the injections.
   Note: Avoid repeated freeze thaw cycles, as virus titer drops significantly between each cycle.
   Note: Adenovirus precipitates are formed by modifying a protocol described previously ³¹.
3. Reconstitute 504 mg of MEM powder with 50 ml of sterile molecular grade water, supplement with 244 mg of sodium bicarbonate and filter in sterile conditions and store at 4 °C.
   1. Prepare the calcium chloride solution by adding 1.5 g of calcium chloride to 50 ml of molecular grade water and filter in sterile conditions and store at 4 °C.
   2. Mix aliquots containing 4 x 10⁸ pfu adenovirus-Cre with sufficient 3% sucrose in sterile water for a final volume of 10 μl. Add 34 µl of MEM to the virus and gently mix. Then add 4 μl of the CaCl₂ solution, gently mix, and incubate at room temperature for 15-20 min.
   3. Store adenovirus on dry ice until ready to form precipitates. Avoid thawing of the adenovirus and storing on ice or room temperature for extended times, unless precipitates are formed.
      Note: It is also possible to mix the sucrose, MEM and CaCl₂ prior to the surgeries if it is not possible to thaw the adenovirus aliquot and begin making precipitates immediately after removal from -80 °C. This aliquot of sucrose, MEM, and calcium can be saved on dry ice until ready to add the adenovirus.
      Note: Virus particles are stable for approximately 1 hr.
   4. Prior to each injection, gently flick the tube to make sure virus particles are mixed. Draw up 3 μl (2.5 x 10⁷ pfu) of virus particles into the 10 μl syringe and prepare the mouse for the intraductal injection.

4. Intraductal Injection of Virus Particles

1. Gently place the mouse on its back onto the illuminated stage of a clean dissection microscope. Illuminate the abdominal side with an extra light source and locate the left 4^th or right 9^th inguinal mammary gland by the small white patches of fur (visible on C57BL/6 females) surrounding each nipple.
2. Rub the nipple gently with a sterile ethanol soaked cotton tipped applicator to clear hair away from the nipple and to sterilize the injection site. If they are difficult to locate, gently apply a thick layer of a depilatory cream or use shears to expose the nipples.
3. Remove the keratin plug, a layer of dense dead skin cells, which is covering the nipple. Once the nipple is exposed, the keratin plug should be easily visible under the dissection microscope.
4. Secure the nipple with fine surgical forceps and pull up with light force to remove the keratin plug.
5. Stabilize the nipple between the forceps.
6. Gently insert the needle between the forceps, cannulating the duct canal at 90°. Enter the nipple slightly past the bevel of the needle (not more than 2 mm) to prevent penetration through the mammary tissue and into the serous membranes of the ventral body cavity.
   1. Do not insert the needle too deep. To ensure proper depth of injection, gently pull the needle up after inserting it into the lumen of the duct, drawing the nipple up along the edges of the needle as it is pulled up.
      Note: Visualization of the injection is difficult; therefore practice for this step is recommended using trypan blue.
7. When the needle is appropriately placed into the mammary duct, release the 3 µl of virus precipitates (2.5 x 10⁷ pfu of adenovirus-Cre) by gently plunging the syringe with the thumb of the hand holding the syringe. The nipple should slightly inflate as the liquid is added.

5. Recovery of Mice

1. Place the mouse back onto the heating pad after the injection until it begins to recover from the anesthesia.
2. Once the mouse is recovered, place it back into a clean cage and monitor for full recovery and movement.
3. 24 hr after the intraductal injection, subcutaneously administer meloxicam at 1 mg/kg.

6. Monitoring Tumor Progression

1. Palpate the injected mammary gland at day 30 for enlargement and swelling.
   1. Monitor tumor progression every 5-7 days once a swollen and enlarged mammary gland is observed.
2. Measure tumor volumes every 3-4 days for tumor growth kinetics once palpable tumors appear (approximately 50-60 days post adenoviral injection).
3. Euthanize mice when tumor volumes exceed 10% of the body weight of the mice.