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 of selected clones led to the isolation of genes which were specific to the ear. Sequence analysis of randomly chosen clones revealed the presence of a histidine rich Ca2+ binding protein, human dynamin, collagen type 1A1, collagen type 2A1, SPARC, human growth hormone, and several specific genes which had no sequence homology in the data base. Furthermore, to apply these techniques for isolating genes specific to distinct inner ear structures and/or cell types of inner ear for which the starting tissue material is limiting, we have used a modified PCR based protocol to construct representative cDNA libraries. We have characterized a cDNA library constructed from small amounts of inner ear tissues recovered by ablative surgical procedure involving labyrinthectomy. The potential application of these protocols for isolating genes involved in hearing and deafness is discussed.
Jacob
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AN
Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Baskaran
N
N
Kandpal
G
G
Narayan
D
D
Bhargava
A K
AK
Kandpal
R P
RP
D000328:Adult; D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification*; D007758:Ear, Inner / Q000737:chemistry / Q000378:metabolism* / Q000601:surgery; D005333:Fetus; D015723:Gene Library; D006801:Humans; D051379:Mice; D011061:Poly A / Q000737:chemistry / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D011897:Random Allocation; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid
D018076:DNA, Complementary; D011061:Poly A