Background The muscle layers of murine gastric fundus have no interstitial cells of Cajal at the level of the myenteric plexus and only possess intramuscular interstitial cells and this tissue does not generate electric slow waves. mice. Fshr Eleven transcripts experienced 2.0C2.5 fold higher mRNA expression in W/WV gastric fundus when compared to wild type tissues. Ten transcripts experienced 2.1C3.9 fold lesser expression in W/WV mutants in comparison with wild type animals. None of these genes have ever been implicated in any bowel motility function. Conclusions These data provides evidence that several important genes have significantly changed in the murine fundus of W/WV mutants that lack intramuscular interstitial cells of Cajal and have reduced enteric motor neurotransmission. Background Interstitial cells of Cajal (ICC) are gastrointestinal (GI) pacemaker cells and intermediaries in enteric motor neurotransmission in the GI tract [1,2]. ICC express KIT/c-kit and depend on signalling via the gene product protein, KIT, a receptor tyrosine kinase which is essential for development and maintenance of the ICC phenotype . Mutations in the white-spotting locus (i.e. W/WV) result in reduced KIT expression. These mutant animals develop few ICC at the level of the myenteric plexus (IC-MY) in the small intestine and the reduced ICC numbers is usually associated with a loss of slow wave activity. Intramuscular ICC (IC-IM) located in the belly, lower esophageal and pyloric sphincters are absent in W/WV mutant animals . Reduced numbers of ICC have also been reported in several GI motility disorders, such as chronic intestinal pseudo-obstruction [5,6], infantile hypertrophic pyloric stenosis [7-9], Hirschsprung’s disease [10-12], slow-transit constipation and certain forms of gastroparesis [13,14]. The association between motility disorders and loss of specific populations of ICC suggests that a more total understanding of the molecular and cell biology of Anacardic Acid IC50 ICC networks within the gastrointestinal tract may help in understanding the etiology of some GI motor pathologies. The aim of the present study was to characterize genetic sequences that Anacardic Acid IC50 are expressed in ICC of the belly that may encode important functional elements of the GI pacemaker/motor neurotransmission system. We pursued the hypothesis that such genes might show differential expression in the small intestines of wild type mice and W/WV mice [15,16]. We previously recognized fifteen known and novel genes that were differentially expressed in the small intestines of wild type and W/WV mice, which develop few IC-MY using a differential gene expression method [17,18]. In the present study we hypothesized that there may also be differential expression of genes in the gastric fundus of W/WV mice, where IC-IM are lost. Our gene microarray analysis successfully recognized 21 genes that were differentially expressed in the fundus of W/WV mice. The differential expression of these mice was confirmed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Methods Animals and Tissue Preparation The use and treatment of experimental animals was approved by the Guideline governing Animal Experiment Committee at the University or college of Yamanashi School of Medicine and at the University or college of Nevada School of Medicine. Six adult male WBB6F1-+/+ mice (wild type) and age matched six adult male WBB6F1-W/WV mice, weighing 20 to 30 g, were purchased from Japan SLC Inc (Shizuoka, Japan). They were anesthetized with ether or carbon dioxide inhalation and sacrificed by cervical dislocation. For gene analyses, stomachs were removed from the animal and the mucosa with the attached submucosa rapidly sharp dissected from your tunica muscularis. Tissues were subsequently frozen in liquid nitrogen (-196 C). Tissues were stored at -80 C until isolation of RNA was preformed . RNA Preparation and Microarray Data Analysis For gene microarray analysis, poly (A)+ RNA was isolated from each tissue sample by TRIZOL (Life Technologies, Inc., Gaithersburg, MD, USA) and Poly (A)+Isolation Kit from Total RNA (ISOGEN, Nippon Gene, Tokyo, Japan).18 Before starting microarray analysis, Anacardic Acid IC50 differential gene expression of KIT between the gastric fundic tissues of wild type mice and those of W/WV mutant mice were confirmed by semi-quantitative RT-PCR (See the section of Semi-quantitative RT-PCR) (Fig. ?(Fig.1A).1A). Microarrays were hybridized and scanned, and image analysis was performed as explained previously [19,20]. In brief, alterations in gene expression were evaluated by reverse transcription of poly (A)+ Anacardic Acid IC50 RNAs in the presence of Cy3 or Cy5 fluorochromes followed by hybridization to mouse GEM 1 microarray chips (Incyte Genomics, Inc., Porter Drive Palo Alto, CA, USA). To confirm the validity of the assay, the experiments were performed in duplicate. The 8734 cDNAs we used represent 7854 unique genes/UniGene clusters: 3205 are annotated and 4649 are unannotated sequences. We assigned a cut-off value, using a variance analysis, to the microarray slide. Genes whose Cy3- and Cy5-transmission intensities were lower than.