Identification of differentially expressed cDNA clones from gerbil cochlear outer hair cells.
In order to identify genes that are associated with outer hair cell(OHC)-specific function, a plasmid library enriched with OHC-specific gene products was constructed using single cell-type-specific complementary DNA (cDNA) and a PCR subtractive hybridization strategy. As a first step, we created separate OHC and inner hair cell (IHC) cDNA pools from individually collected cells using a nonspecific reverse transcription polymerase chain reaction. Next, the OHC cDNA was subtracted against IHC cDNA using a PCR-based subtractive technique. IHCs and OHCs share many common features, making IHC cDNA an ideal 'driver' to 'subtract away' common hair cell gene products and enrich differentially expressed cDNAs, including OHC-specific genes. The subtracted OHC cDNAs were then cloned to generate an OHC - IHC subtracted cDNA plasmid library. Finally, a differential screening procedure was performed, resulting in 477 differentially positive clones. After analysis of these 477 clones, 50 known genes were identified, including two previously known OHC-specific proteins: oncomodulin and the recently described motor protein prestin. An additional 84 novel clones were also found. As this library of cDNA fragments represents differentially expressed genes in OHCs, it can be used as starting material for isolation and characterization of a complete set of OHC gene products, an important step in investigating normal and abnormal cochlear function.
Zheng
Jing
J
Auditory Physiology Laboratory (Hugh Knowles Center), Department of Communication Sciences and Disorders, Northwestern University, Evanston, IL 60802, USA. jzh215@northwestern.edu
Long
Kevin B
KB
Robison
Donald E
DE
He
David Z Z
DZ
Cheng
Jennifer
J
Dallos
Peter
P
Madison
Laird D
LD
D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002999:Clone Cells; D003051:Cochlea / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics*; D003638:Deafness / Q000235:genetics* / Q000378:metabolism; D005849:Gerbillinae; D006199:Hair Cells, Auditory, Inner / Q000378:metabolism; D018072:Hair Cells, Auditory, Outer / Q000378:metabolism*; D009693:Nucleic Acid Hybridization; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D024661:Potassium Channels, Inwardly Rectifying / Q000378:metabolism; D011506:Proteins / Q000235:genetics
D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D024661:Potassium Channels, Inwardly Rectifying; D011506:Proteins; C034733:oncomodulin