Gene expression changes in FORKO mice

To understand consequences of loss of FSH receptor, we used genetically altered mouse mutants with deletion of this receptor. Follitropin Receptor Knockout (FORKO) mice present various developmental and age-dependent abnormalities such as infertility, senescence and increased tumor incidences in addition to other phenotypes. To explore why the atrophic ovaries of FORKO mice develop ovarian anomalies later in life, we used expression profiling studies to gain a more comprehensive view of genes that are misregulated. Keywords: Comparison of Wildtype and FSH-Receptor Knock out Ovaries at 8 months of age. Mice were housed under controlled temperature and constant light (12 h of light, 12 h of darkness), with food and water provided ad libitum. The female mice used in this experiment were derived by breeding heterozygotes of the 129T2 svEmsJ background. For RNA extraction, both the ovaries from 8 month old mice were carefully dissected and pooled. Tissues were frozen in liquid nitrogen and homogenized in TRIZOL (Invitrogen, Burlington, Ontario) and processed in accordance with the manufacturer’s protocol. Affymetrix 39 K GeneChip® Mouse Genome 430 2.0 Array has over 45000 probe sets which analyses 39000+ transcripts and variants from over 34000 well characterized mouse genes. A total of 1 µg of double-stranded cDNA was transcribed in vitro using the Bioarray High-Yield RNA Transcript Labeling Kit (Enzo, Farmingdale, NY) according to the manufacturer's instructions using biotinylated CTP and UTP (Enzo, Farmingdale, NY). Following 5-hr incubation at 37°C, the resultant biotin-labeled cRNA was purified with RNAeasy columns (Qiagen) and eluted in 40 µl of RNAse-free water. The concentration of biotin-labeled cRNA was determined by RNA bio-analyzer. Target cRNAs corresponding to either wild type ovary or FORKO ovarian tissue were hybridized to an individual GeneChip from an identical lot of Affymetrix 39 K GeneChip® Mouse Genome 430 2.0 array for 16 hr. GeneChip arrays were washed and stained using antibody-mediated signal amplification and the Affymetrix Fluidics Station's standard Eukaryotic GE Wash 2 protocol, using Affymetrix equipment and protocols (Affymetrix, Santa Clara, CA).