Neuroscience Letters 482 (2010) 230–234 Contents lists available at ScienceDirect Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet Cloning of expressed sequence tags (ESTs) representing putative epileptogenesis-related genes and the localization of their expression in the normal brain Marzena Stefaniuk, Katarzyna Lukasiuk ∗ Epileptogenesis Lab, The Nencki Institute of Experimental Biology, 3 Pasteur Str, 02-093 Warsaw, Poland article info Article history: Received 18 May 2010 Received in revised form 13 July 2010 Accepted 19 July 2010 Keywords: Brain Methyl-CpG binding domain protein 3 Selenoprotein T Sorting nexin-2 Transmembrane protein 204 Tweety homolog 1 abstract The goal of this study was to clone and sequence selected expressed sequence tags (ESTs) which mRNA is upregulated in a rat model of epilepsy based on our previous work [17] and to determine the local- ization of their mRNA expression in the normal brain. Six ESTs that had not been assigned to any gene at the time of the inception of our experiments were chosen for analysis. Radioactive in situ hybridiza- tion revealed that the expression of four transcripts (AA955087, AA875438, AA899079, AA819660) was clearly localized to hippocampal neurons and was also detected in the cortex. Two transcripts, AA819523 and AA819766, displayed a uniform expression pattern. 5  RACE cloning and sequencing allowed for the annotation of five ESTs to known or predicted genes: sorting nexin-2 (SNX2), tweety homolog 1 (Ttyh1), selenoprotein T (SelT), transmembrane protein 204 (Tmem204) and methyl-CpG binding domain protein 3 (Mbd3). According to the results of our combined non-radioactive in situ hybridization and immuno- histochemistry with cell-specific markers, mRNA coding for Ttyh1, Tmem204 and Mbd3 was expressed in neurons. The potential role of the studied genes in normal brain or in brain pathology remains elusive and requires further study because little is known about these genes in general. © 2010 Elsevier Ireland Ltd. All rights reserved. Expressed sequence tags (ESTs) are fragments of mRNA sequences derived through single sequencing reactions performed on ran- domly selected clones from cDNA libraries. ESTs are generated mainly for gene discovery or in genome projects. To date, over 45 million ESTs have been generated [26]. A typical EST sequence is only 200–800 bp in length, and, because ESTs are sequenced only once, the sequence is prone to errors [1,24]. The largest source of EST sequences is dbEST, a division of GeneBank run by NCBI (http://www.ncbi.nlm.nih.gov). Currently, the dbEST database con- tains sequences of over 65 million ESTs from different organisms, including 8.3 million human, 4.8 million mouse and over 1 mil- lion rat ESTs. On the basis of their sequence, ESTs can be grouped to single clusters that tag transcripts of the same gene. These gene clusters can be annotated to the known gene. Because ESTs can also represent unknown genes, they can assist in the discovery of new genes. ESTs are frequently used as probes in microarrays. Because microarrays enable the evaluation of the expression levels of many thousands of genes simultaneously, they are increas- ingly popular in studies of gene expression. Microarrays have been successfully employed in the detection of alterations in gene expression patterns in psychiatric diseases, brain devel- ∗ Corresponding author. Tel.: +48 22 5892434. E-mail address: k.lukasiuk@nencki.gov.pl (K. Lukasiuk). opment, brain tumors, neurodegenerative diseases and epilepsy [5,7,17,18,21,22]. In epilepsy research, studies of alterations in gene expression at the level of whole transcriptomes with microarrays led to the discovery of new molecular events critical for disease development, including inflammatory and immune responses, the complement cascade, proteolysis and TGF- (Transforming Growth Factor beta) signaling [3,9,16,18]. These pathways could serve as targets for the design of new antiepileptic or antiepileptogenic therapies. However, the relevance of alterations in the expres- sion of many other genes detected in these studies is not known, especially in the case of genes represented on microarrays by ESTs. In our previous microarray study, we found changes in the expression levels of a number of ESTs representing genes of unknown identity during epileptogenesis and in epilepsy using an animal model of temporal lobe epilepsy [17]. The present study was designed to clone and determine the expression of six such ESTs in the normal brain. In all experiments, adult male Sprague–Dawley rats (290–360 g) were used (Institute of Biochemistry and Biocybernetics, War- saw, Poland). All experiments were approved by the Animal Care and Use Committee. In the handling and care of all ani- mals, the guidelines from the European Union Council Directive 86/609/EEC for animal research were strictly followed. Animals were housed in controlled environmental conditions with access 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.07.045