ISOLATION OF HUMAN EAR SPECIFIC CDNAS AND CONSTRUCTION OF CDNA LIBRARIES FROM SURGICALLY REMOVED SMALL AMOUNTS OF INNER-EAR TISSUES

We have used representational difference analysis (RDA)for subtractive hybridization of oligo dT primed directionally cloned cDNA libraries from human inner ear tissue and a B-lymphoblast cell line, Two rounds Of subtraction-amplification, followed by differential hybridization o f selected clones led to the isolation of genes which were specific to the ear. Sequence analysis of randomly chosen clones revealed the pre sence of a histidine rich Ca2+ binding protein, human dynamin, collage n type 1A1, collagen type 2A1, SPARC, human growth hormone, and severa l specific genes which had no sequence homolog in the delta base. Furt hermore, to apply these techniques for isolating genes specific to dis tinct inner-ear structures and/or cell types of inner ear for which th e starting tissue material is limiting, we have used a modified PCR ba sed protocol to construct representative cDNA libraries. We have chara cterized a cDNA library constructed from small amounts of inner ear ti ssues recovered by ablative surgical procedure involving labyrinthecto my. The potential application of these protocols for isolating genes i nvolved in hearing and deafness is discussed.