Yamamoto et al. BMC Genomics 2012, 13:335 http://www.biomedcentral.com/1471-2164/13/335 Page 3 of 15 Figure 1 Overview of the experimental procedure for bacterial reconstitution. During the experiment, all mice (including SPF mice) were housed in a Trexler-type flexible film isolator in a standard germ-free state. For generation of 0WexGF (mice, i.e., GF mice with bacterial reconstitution at the time of delivery), pregnant GF mice were housed with SPF female mice 1 day before delivery and only male pups were retrieved after weaning. To generate 5WexGF mice (i.e., GF mice with bacterial reconstitution at 5 weeks old), since 5 weeks, GF male mice began to cohabit with SPF female mice of the same age. Male mice in all groups were sacrificed at 9 weeks of age. and LIV in addition to the intestines. SPL is a key part of body’s immune system and, therefore, comparison of the gene expression profiles of SPL and intestines may help to elucidate the cross-talk between intestinal local immunity and general immunity. The gene expression profile of LIV enzymes, especially those of steroid and xenobiotic metabolism, has been shown to be pro- foundly affected by intestinal microflora via nutrients and metabolites transported through portal vein [38]. Further, translocation of bacteria-derived substances, e.g., lipopolysaccharide to the LIV, is also known to have a great impact on the host immune system. GO analysis: overview and gene clustering Firstly, we have summarized the biological impact of en- teric microbiota on host organs by MGP analysis on GO (http://www.geneontology.org/) categories. Figure 2 shows Venn diagrams of the numbers of differentially expressed genes and overrepresented GO categories of Biological Process (BP). Results from 3 comparisons (SPF vs. GF, 0WexGF vs. GF, and 5WexGF vs. GF) revealed alteration of an extremely large number of genes (probe sets) in the SI of SPF mice compared with those of 0WexGF and 5WexGF mice. There were approximately 22 to 36 com- monly regulated probe sets in the LI, SI, LIV, and SPL among the 3 colonization models. At least 300 probe sets were listed in each group in the LI, SI, and SPL; however, no or only a small number of GO categories were overre- presented in most cases with the exception of LI in 0WexGF mice, SI in SPF mice, and SPL in 5WexGF mice. The overrepresented GO categories are listed in Table 1 (LI of 0WexGF), Table 2 (SI of SPF) and Table 3 (SPL of 5WexGF). Specific alteration of genes for antigen presen- tation in LI of 0exGF mice, and for energy cycle and ner- vous development in SI of SPF mice, were identified from these tables. The results of unsupervised hierarchical cluster ana- lysis of gene expression patterns are shown in Figure 3, Figure 4, Figure 5 and Figure 6. The clusters denoting overrepresented GO categories included immune system development/B cell activation/hematopoiesis (in LI) and metabolic process/lipid metabolic process (in SI). Analysis of signal pathway Next, in order to gain information regarding the mo- lecular events evoked by enteric microbiota, we applied