Akt1 in Mammary Development Akt1 deficiency also decreased the total number of alveolar buds and ducts during puberty (PND 50), which persisted into adulthood (PND 70) (Fig. 2 J). While Akt12/2 glands eventually reach the same size as their wild-type counterparts, the overall alveolar bud and terminal duct population remains lower, indicating a permanent change in mammary gland structure. This alveolar bud deficiency could be attributed to the earlier lack of terminal end buds, which contain the progenitor cells for alveolar and ductal myoepithelial and epithelial cells. Importantly, this apparent lack of alveolar buds and ducts may be a potential reason for abnormal mammary gland reconstruction during pregnancy and lactation in Akt12/2 glands as seen in other studies [14]. Akt1 influences mRNA patterns in the adult mammary gland The deficiency in alveolar buds and terminal ducts in Akt12/2 adult mammary glands led us to examine the effects of Akt1 deficiency on gene expression in the adult mammary gland. We performed Affymetrix gene arrays on PND 70 Akt1+/+ and Akt12/2 thoracic mammary glands, which indicated differential expression of 119 genes with a fold change of ,1.5 and a p-value of ,0.05 (Table S1). Real-time RT-PCR validation of selected genes from the gene array indicated strong correlation between gene array results and RT-PCR. The genes chosen for validation were butyrophilin, subfamily 1, member A1 (Btn1a1), v-akt murine thymoma viral oncogene homolog 1 (Akt1), myotubularin related protein 7 (Mtmr7), Figure 3. Akt1 influences gene expression patterns in the adult mammary gland. Microarray results were validated with qRT-PCR for the genes Btn1a, Akt1, Mtmr7, Muc1, Lbh, Dsc3, Pou2af, and Faim3. Data represented as fold change in gene expression for Akt12/2 vs. Akt1+/+ (A). IPA analysis generated the top ten biological functions and diseases altered by the loss of Akt1 (B) IPA analysis generated an association network connecting cardiovascular system development and function, organismal development, and cancer (C). Statistical analyses for qRT-PCR were conducted using a two-tailed student’s t test. **p,0.01, *p,0.05, #p,0.1. n = 3 per genotype for microarray, n = 6 per genotype for qRT-PCR. doi:10.1371/journal.pone.0024432.g003 PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e24432