BMC Genomics 2006, 7:42 http://www.biomedcentral.com/1471-2164/7/42 Table 1: Genes more highly expressed in the duodenum of genetically susceptible animals compared to resistant Human Refseq: Genbank accession number Gene description NM_003226* Intestinal trefoil factor 3 NM_006398* Ubiquitin D NM_003122* Serine protease inhibitor, Kazal type 1 NM_001009555 SH3 domain protein D19 NM_004295 TNF receptor-associated factor 4 NM_022342 Kinesin family member 9 NM_005646 TAR (HIV) RNA binding protein 1 NM_033503 Bcl2 modifying factor NM_003392 Wingless-type MMTV integration site family, member 5A NM_000179 MutS homologue 6 NM_001655 Archain 1 NM_079423 Myosin light polypeptide 6 NM_006184 Nucleobindin 1 NM_178156 Fucosyltransferase 8 NM_005533 Interferon induced protein 35 NM_000062 Serine (or cysteine) proteinase inhibitor, clade G NM_002133 Decycling heme oxygenase 1 NM_002816 Proteasome 26S subunit NM_002116 Major histocompatibility complex, class I, A NM_013442 Stomatin-like 2 NM_201265 Bone marrow stromal cell-derived ubiquitin-like 7 NM_016248 A kinase anchor protein 11 NM_006808 Sec61 beta subunit NM_032492 Jagunal homologue 1 NM_002083 Glutathione peroxidase 2 Gene name TFF3 UBD SPINK1 EVE1 TRAF4 KIF9 TARBP1 BMF WNT5A MSH6 ARCN1 MYL6 NUCB1 FUT8 IFI35 SERPING1 HMOX1 PSMD12 HLA-A STOML2 BMSC-UbP AKAP11 SEC61B JAGN1 GPX2 Ensembl human gene Cytogenetic band 21q22.3 6p22.1 5q32 4q31.3 17q11.2 3p21.31 1q42.2 15q15.1 3p14.3 2p16.3 11q23.3 12q13.2 19q13.33 14q23.3 17q21.31 11q12.1 22q12.3 17q24.2 6p21.33 9p13.3 15q24.1 13q14.11 9q22.33 3p25.3 14q23.3 Contig BLAST E value 3 × 10-59 1 × 10-52 2 × 10-58 0 0 0 0 1 × 10-18 6 × 10-58 0 0 0 0 0 3 × 10-77 2 × 10-50 0 0 8 × 10-35 0 0 1 × 10-44 1 × 10-138 1 × 10-180 0 Fold increase in expression 1.2 1.3 1.3 1.8 1.1 1.4 1.2 1.2 1.2 1.2 1.2 1.1 1.1 1.3 1.1 1.2 1.1 1.2 1.1 1.2 1.3 1.2 1.1 1.1 1.2 P value 7.7 × 10-20 1.8 × 10-12 5.5 × 10-10 3 × 10-9 1.3 × 10-8 1.8 × 10-7 2 × 10-7 3.7 × 10-7 4.1 × 10-7 8 × 10-7 1.4 × 10-6 1.9 × 10-6 3.4 × 10-6 4.5 × 10-6 6.7 × 10-6 9 × 10-6 1.1 × 10-5 1.1 × 10-5 1.2 × 10-5 1.2 × 10-5 1.5 × 10-5 1.8 × 10-5 1.9 × 10-5 2.8 × 10-5 3.3 × 10-5 * More than one EST corresponding to this gene was differentially expressed and their probabilities were combined. Results The microarray experiment examined gene expression in duodenum tissue from four resistant and four susceptible naïve Perendale lambs. Poly(A)+ RNA isolated from duo- denum tissue was reverse transcribed into cDNA, fluores- cently labelled and hybridised to ovine 20 k cDNA microarrays. The experiment design was a factorial dye swap design involving 16 slides, where every animal was compared to every animal in the opposite selection line, as described previously [25]. One hundred ESTs showed differential expression (Figure 1) between the resistant and susceptible animals. Forty ESTs were more highly expressed in the resistant line while 60 were more highly expressed in the susceptible line. These ESTs were rese- quenced in order to verify the insert sequence. Confirmed ESTs were annotated using BLASTN against the human RefSeq database and a cut-off E value of 1 × 10-18. Thirty of the ESTs more highly expressed in susceptible animals could be verified and annotated in this manner giving 25 unique human RefSeqs. Sixteen of the ESTs more highly expressed in resistant animals could also be annotated. The lists of differentially expressed human RefSeqs is given in Tables 1 and 2. Northern blotting was carried out to confirm differential expression of a number of genes (Figure 2). TFF3 encodes intestinal trefoil factor 3. This gene is a member of a fam- ily of trefoil factors that is involved in mucosal mainte- nance and repair and is known to be overexpressed during inflammatory processes [26]. In accordance with the microarray results, this gene had a 20% increase in expres- sion in the duodenum of the susceptible animals com- pared to the resistant (Figure 2A). While the degree of upregulation was not marked it was still significant (P = 0.05). Differential expression of the pancreatic secretory trypsin inhibitor gene (SPINK1) was also confirmed. SPINK1 encodes a gene also known as TATI (tumour associated trypsin inhibitor) that is expressed mainly in the pancreas but is also expressed in the mucosa of the small intestine [27]. One of the major roles of SPINK1 is thought to be the prevention of premature activation of pancreatic pro- teases. This has the effect of decreasing the rate of mucus digestion by luminal proteases within the stomach and colon. SPINK1 is also known to increase the proliferation of a variety of cell lines and to stimulate cell migration, Page 3 of 12 (page number not for citation purposes)