Deaf-1 regulated genes in the mouse mammary gland

Microarray analysis was used to compare the gene expression profiles of Deaf-1-transduced mouse mammary epithelial cells (MECs) relative to Deaf-1-deficient MECs. To investigate potential target genes of Deaf-1, primary mammary epithelial cells (MECs) were isolated from a Deaf-1-null mouse and immortalised by transduction with a retrovirus encoding the human papilloma virus (HPV16) E6/E7 proteins. Two clones were selected and transduced with either an empty retrovirus or one encoding Deaf-1. RNA was subsequently isolated from cells at 24 and 48 hours after transduction for Affymetrix analysis. High levels of Deaf-1 expression were demonstrated in cells transduced with the Deaf-1 retrovirus relative to controls following a 48 hour selection in puromycin. This time-point was therefore selected for further analysis. To compare the gene expression profiles of Deaf-1-transduced MECs relative to Deaf-1-deficient MECs, Affymetrix analysis was performed using the GeneChip® Mouse Expression Set 430 2.0 array which comprises 39,000 transcripts on a single array. Total RNA was harvested from two independent clones (C1 and E1) infected with either control or Deaf-1-expressing retrovirus making 4 samples in total. Clones C1 and E1 were treated as biological replicates in the subsequent analysis.