Identification of unique transcripts from a mouse full-length, subtracted inner ear cDNA library $ Kirk W. Beisel, a, * Toshiyuki Shiraki, b Ken A. Morris, a Celine Pompeia, c Bechara Kachar, c Takahiro Arakawa, b Hidemasa Bono, b Jun Kawai, b Yoshihide Hayashizaki, b and Piero Carninci b a Department of Biomedical Sciences, Creighton University, 2500 California, Omaha, NE 68178, USA b Laboratory for Genome Exploration Research Group, RIKEN Genomic Sciences Center, Tsukuba, Japan c Section on Structural Cell Biology, NIDCD, National Institutes of Health, Bethesda, MD 20892, USA Received 9 October 2003; accepted 25 January 2004 Available online 9 April 2004 Abstract A small-scale full-length library construction approach was developed to facilitate production of a mouse full-length cDNA encyclopedia representing f250 enriched, normalized, and/or subtracted cDNA libraries. One library produced using this approach was a subtracted adult mouse inner ear cDNA library (sIEa). The average size of the inserts was f2.5 kb, with the majority ranging from 0.5 to 7.0 kb. From this library 22,574 sequence reads were obtained from 15,958 independent clones. Sequencing and chromosomal localization established 5240 clusters, with 1302 clusters being unique and 359 representing new ESTs. Our sIEa library contributed 56.1% of the 7773 nonredundant Unigene clusters associated with the four mouse inner ear libraries in the NCBI dbEST. Based on homologous chromosomal regions between human and mouse, we identified 1018 UniGene clusters associated with the deafness locus critical regions. Of these, 59 clusters were found only in our sIEa library and represented f50% of the identified critical regions. D 2004 Elsevier Inc. All rights reserved. The mammalian inner ear represents a unique sensory organ with a large number of specialized structures and cell types. Both neurosensory/neuronal and nonsensory cells represent the cellular components for the detection and identification of external stimuli [1]. The nonsensory cells can be further subdivided into the following functional groups. These are those involved in: (1) the production of the K + -rich endolymph and the associated K + recirculation [2], (2) the maintenance of a nonporous endolymphatic space, (3) the production and maintenance of extracellular matrix in mantle required for mass loading of the specialized apical apparatus of the neurosensory epithelium [3], (4) the cyto- skeleton architecture of the supporting and the neurosensory cells, and (5) the morphological structure and integrity of the inner ear [4]. The mammalian auditory organ, the cochlea, has been further specialized to detect sound frequencies higher than 10 kHz through utilization of a flexible basilar mem- brane and cochlear amplification by outer hair cells [5–7]. The functions of hearing and balance most likely require a wide variety of genes to develop and maintain the cellular and structural components of the inner ear. A number of different genes are associated with hereditary inner ear disorders. In human there are at least 26 syndromic and 80 nonsyndromic loci associated with inner ear disorders (G. van Camp and R.J. Smith, http://www.uia.ac.be/dnalab/ hhh), of which about 50% of the corresponding genes are known. In mouse at least 175 spontaneous or genetically manipulated mutant mouse lines that carry defective genes causing hearing loss and/or balance dysfunction are identi- fied (K.P. Steel et al., http://www.ihr.mrc.ac.uk/hereditary/ MutantsTable.shtml; Q.Y. Zhang and K. Johnson, http:// www.jax.org/research/hhim/documents/map.html). The discovery of specialized genes in the inner ear has lagged behind the functional genomics of other organs and tissues and has been hindered by relative inaccessibility of the inner ear, limited quantities of available tissue, and the difficulty of culturing many inner ear cell types. The iden- tification, procurement, and construction of full-length cDNA libraries from the inner ear still represent a significant hurdle for the identification of inner ear-pertinent transcripts. 0888-7543/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ygeno.2004.01.006 $ Supplementary data for this article may be found on ScienceDirect. * Corresponding author. Fax: +1-402-280-2690. E-mail address: beisel@creighton.edu (K.W. Beisel). www.elsevier.com/locate/ygeno Genomics 83 (2004) 1012 – 1023