Doxycycline hyclate chow

Autochthonous tumor generation: KP-NINJA mice were infected intratracheally with 2.5 × 10⁷ PFU Ad5mSPC-Cre (Dr. Anton Berns, Netherlands Cancer Institute), after precipitation with 10mM CaCl₂ for 20–60 minutes, or 5 × 10⁴ PFU Lenti-cre. To induce expression of NINJA neoantigen in infected cells, mice were given doxycycline hyclate chow (625mg/kg; Envigo cat. TD.09628) days 7–11 post infection (p.i.) and concomitantly treated with 4.4mg tamoxifen (MP Biomedicals cat. MP215673894) in corn oil (ThermoFisher Scientific cat. S25271) by gavage on days 8–10 p.i. To induce neoantigen expression via lentivirus, KP mice were infected with 2.5 × 10⁴ PFU mClover-GP33–80-Cre lentivirus and assessed at 8 weeks p.i. Orthotopic KPN1 tumor transplants: Established KPN1 cells were maintained in complete DMEM (10% HI-FBS, 55μM beta-mercaptoethanol, 1x Pen/Strep and 1x L-Glut). Prior to injection, cells were washed 3x with 1xPBS and 200,000 cells were injected intravenously via tail vein injection. Subcutaneous KPN1 transplants: Established KPN1 cells, sorted for GFP⁺ (NINJA-expressing) cells, were maintained in complete DMEM (10% HI-FBS, 55μM beta-mercaptoethanol, 1x Pen/Strep and 1x L-Glut). Prior to injection, cells were washed 3x with 1xPBS and 500,000 cells were injected s.c. and measured using standard caliper measurements. Tumor volume = (LxW²)/2.

TRE-Ccnd1^WT and TRE-Ccnd1^K112E mice transgenic for human Ccnd1 with or without the K112E mutation under TRE regulation have been published previously^{31 (link),32 (link)}. Pax7^rtTA mice contain rtTA-M2 followed by an IRES, mouse Pax7, and polyadenylation tail knocked in to the first exon of the Pax7 locus. Both lines were maintained by breeding heterozygous animals to wild-type animals. Experimental animals (Pax7^rtTA/+, Pax7^rtTA/+; TRE-Ccnd1^WT, and Pax7^rtTA/+; TRE-Ccnd1^K112E) were generated by intercrossing the two lines and aging the appropriate genotypes until use. All transgenic animals were treated for seven days with doxycycline by providing doxycycline hyclate chow (Envigo, TD.120769), delivering a daily dose of 2-3 mg doxycycline based on consumption of 4-5 grams each day. On the sixth and seventh days, doxycycline hyclate (Thermo Fisher ICN19895510) was injected intraperitoneally at 50 mg/kg in 0.9% NaCl. MuSCs were isolated on the eighth day based on immunophenotype via FACS as described above.