Fvb n mice

Mammary glands from 5 to 15 (depending on the experiment) wild type 8–10 week old FVB/N mice (Jackson Laboratory) were harvested, minced, digested, trypsinized, and lineage depleted as detailed in the flow cytometry methods section. For total population reconstitution, lineage positive depleted cells were infected for 2 hours with an MOI of 20 of either pHIV-ZSG empty vector control (Addgene, #18121) or pHIV-ZSG CD8-IGF1R containing virus under centrifugation in ultra low attachment plates and implanted, same-day, into cleared fat pads of 23 day old recipient mice as previously described [40 ]. For reconstitution of sorted populations, cells were sorted for mammary lineages as described in flow cytometry methods and then infected overnight at 37° in ultra-low attachment plates. The next morning, cells were collected. Fat pads were cleared from 23-day old FVB/N mice (Jackson Laboratory) [40 ]. Using a Hamilton 25ul syringe, 10ul of media containing 4,000 to 15,000 cells, depending on the experiment, was injected into the cleared fat pad. Mice were left for 8 weeks to allow the gland to reconstitute. Glands were harvested, analyzed for green fluorescence and either carmine stained for analysis of reconstitution and/or prepared into FFPE blocks.

All animal procedures were approved by the Institutional Animal Care and Use Committee at University at Buffalo, The State University of New York. Age-matched male FVB/N mice (25–35 g; The Jackson Laboratory, Bar Harbor, ME, USA) were housed in an animal care facility maintained at 20 ± 2.0°C on a 12-h light/dark cycle (lights on from 6 AM to 6 PM) with ad libitum food and water.