Gentra puregene protocol

Female CMV-Cre mice (Jax strain 006054)^{45 (link)} were crossed with the Rosa26::LSL-A3B^{16 (link)} and Rosa26::LSL-CAG-A3B (these studies) animals. For mammary ductal cell-specific expression of A3B, Rosa26::LSL-CAG-A3B mice were crossed with MMTV-Cre mice (Jax strain 003551). The resulting pups were genotyped, enrolled, and monitored weekly and aged out until tumors could be observed either visually or by palpation. Genomic DNA was isolated using the Gentra Puregene protocol (Qiagen) on mouse tail biopsies from animals at 21 days of age, with 50 ng of DNA used as a PCR template. A genotyping schematic for Rosa26::LSL-A3B and Rosa26::LSL-CAG-A3B mice is provided in Figure S1. MMTV-Cre mice were genotyped for Cre using RSH17507 and RSH17508. Mice were monitored three times a week for signs of excessive pain or discomfort, or until their tumors reached >1 cm.^{3 (link)} All mice were euthanized via CO₂ asphyxiation, then control tissues and tumors were immediately collected to be fixed in buffered 10% formalin or flash-frozen in liquid nitrogen. Tumors were initially scored based on visual diagnosis, and then subsequently confirmed with histopathological analysis.

DNA was extracted from fetal or placental tissue, using the Gentra PureGene protocol (Qiagen, Hilden, Germany). DNA concentrations were measured using a Qubit fluorometer together with the DS BR DNA assay (Thermo Fisher Scientific, Waltham, MA, USA). DNA quality was assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific), where A260/A280 ratios between 1.8 and 2.0, and A260/A230 ratios >1.5 were accepted. DNA fragmentation was evaluated using agarose gel electrophoresis (1.5% agarose).