Molecular cloning of the Coch gene of guinea pig inner ear and its expression analysis in cultured fibrocytes of the spiral ligament

• Published in: Acta oto-laryngologica Vol. 130; no. 8; pp. 868 - 880
• Main Authors: Li, Lishu, Ikezono, Tetsuo, Sekine, Kuwon, Shindo, Susumu, Matsumura, Tomohiro, Pawankar, Ruby, Ichimiya, Issei, Yagi, Toshiaki
• Format: Journal Article
• Language: English
• Published: Stockholm Informa UK Ltd 01.08.2010; Taylor & Francis; Informa
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Biological and medical sciences; Blotting, Western; Cattle; Cells, Cultured; Cloning, Molecular; Cochlin; culture; cytokine; Cytokines - metabolism; Disease Models, Animal; DNA, Complementary - chemistry; Extracellular Matrix Proteins; Female; Guinea Pigs - genetics; Guinea Pigs - metabolism; Humans; Immunohistochemistry; Medical sciences; Molecular Sequence Data; Otorhinolaryngology. Stomatology; Protein Isoforms - metabolism; Proteins - genetics; Proteins - metabolism; RNA, Messenger - metabolism; Sequence Homology, Amino Acid; Spiral Ligament of Cochlea - cytology; Spiral Ligament of Cochlea - metabolism; Cell culture; Fibrocyte; Rodentia; Cytokine; ENT; Inner ear; Cochlin; Vertebrata; Mammalia; Guinea pig; Gene; Spiral ligament; Animal; culture; Genetics; Molecular biology; Molecular cloning
• ISSN: 0001-6489; 1651-2251
• DOI: 10.3109/00016480903493766

Abstract Conclusions: We have cloned guinea pig Coch cDNA and the sequence information will be useful for future molecular study combined with physiological experiments. Proper Coch gene expression appears to be dependent on the unique extracellular micro-environment of the inner ear in vivo. These results provide insight into the Coch gene expression and its regulation. Objective: To characterize the guinea pig Coch gene, we performed molecular cloning and expression analysis in the inner ear and cultured fibrocytes of the spiral ligament. Methods: The Coch cDNA was isolated using RACE. Cochlin isofoms were studied by Western blot using three different types of mammalian inner ear. The cochlear fibrocytes were cultured and characterized by immunostaining. Coch gene expression in the fibrocytes was investigated and the influence of cytokine stimulation was evaluated. Results: The full-length 1991 bp Coch cDNA that encodes a 553 amino acid protein was isolated. The sequence had significant homology with other mammals, and the sizes of the Cochlin isoforms were identical. In the cultured fibrocytes, Coch mRNA was expressed in a very small amount and the isoform production was different, compared with the results in vivo. Cytokine stimulation did not alter the level of mRNA expression or isoform formation.