ABSTRACT: In this study we used the cre-loxP recombinase system. All mice were genetically modified in as much as all mice had exon 4 of the IGF-I gene flanked by loxP sites. In the knockouts, Mx-Cre had also been introduced, which results in inactivation of the IGF-I gene specifically in the liver. RNA from liver, skeletal muscle, heart, fat (retroperitoneal fat) and bone (vertebrae) was extracted from 6 control and 7 mice deficient of liver-derived insulin-like growth factor-I (LI-IGF-I-/- mice) by Tri Reagent (Sigma, St. Louis, MO, USA) and purified using spin columns from Rneasy Total RNA Isolation Kit (Qiagen, Chatsworth, CA, USA). For microarray analysis, the RNA samples were pooled three or two, resulting in three pools per group while for the confirmatory RT-PCR analyses individual mice (n=6-7) were analyzed. For each organ, there were three pooled samples from the mice with deficiency of liver-derived IGF-I and 3 pooled samples from the wildtype mice i.e. intact IGF-I gene. The pooled RNA was reverse transcribed into cDNA, labeled, and analyzed by DNA microarray (MG-U74Av2 Array; Affymetrix, Santa Clara, CA, USA). Scanned output files were analyzed using Affymetrix Micro Array Suite version 4.0.1 software (Affymetrix). To allow comparison of gene expression, the gene chips were globally scaled to an average intensity of 500. Each of the three LI-IGF-I-/- chips was compared with the three control chips, generating a total of nine comparison files. Only the genes that were regarded as M-^SchangedM-^T according to the Affymetrix algorithm in four to nine of the comparisons were selected for further analysis. We then calculated an average-fold change for the nine comparisons of the selected genes. For a gene to be regarded as consistently regulated in the LI-IGF-I-/- mice, the average-fold increase or decrease of the nine comparisons was set as at least 1.5 fold in at least 4 of the 5 analyzed organs. Microarray showed that heat shock protein 1A (Hspa1a), heat shock protein 1B (Hspa1b), and connective tissue growth factor (Ctgf) were decreased in all organs that expressed these genes. CCAAT/enhancer protein delta (Cebpd) decreased in 4 of the 5 analyzed organs. The RT-PCR analyses that were performed later confirmed that mRNA levels of Hspa1a, Hspa1b, and Ctgf decreased in LI-IGF-I-/- mice in skeletal muscle tissue while mRNA levels of Cebpd were unchanged.
