Identification of differentially expressed cDNA clones from gerbil cochlear outer hair cells

Jing Zheng, Kevin B. Long, Donald E. Robison, David Z.Z. He, Jennifer Cheng, Peter Dallos, Laird D. Madison

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)277-288
Number of pages12
JournalAudiology and Neuro-Otology
Volume7
Issue number5
DOIs
StatePublished - Oct 9 2002

Fingerprint

Outer Auditory Hair Cells
Gerbillinae
Inner Auditory Hair Cells
Complementary DNA
Clone Cells
Genes
Polymerase Chain Reaction
Plasmids
Cochlea
Gene Library
Reverse Transcription

Keywords

  • COLIA2
  • Inner hair cells (IHC)
  • Kir7.1
  • Nonsyndromic deafness loci
  • OHC-specific genes
  • Oncomodulin
  • Outer hair cells (OHC)
  • Prestin
  • Subtracted library

ASJC Scopus subject areas

  • Physiology
  • Otorhinolaryngology
  • Sensory Systems
  • Speech and Hearing

Cite this

Zheng, J., Long, K. B., Robison, D. E., He, D. Z. Z., Cheng, J., Dallos, P., & Madison, L. D. (2002). Identification of differentially expressed cDNA clones from gerbil cochlear outer hair cells. Audiology and Neuro-Otology, 7(5), 277-288. https://doi.org/10.1159/000064443
Zheng, Jing ; Long, Kevin B. ; Robison, Donald E. ; He, David Z.Z. ; Cheng, Jennifer ; Dallos, Peter ; Madison, Laird D. / Identification of differentially expressed cDNA clones from gerbil cochlear outer hair cells. In: Audiology and Neuro-Otology. 2002 ; Vol. 7, No. 5. pp. 277-288.
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Zheng, J, Long, KB, Robison, DE, He, DZZ, Cheng, J, Dallos, P & Madison, LD 2002, 'Identification of differentially expressed cDNA clones from gerbil cochlear outer hair cells', Audiology and Neuro-Otology, vol. 7, no. 5, pp. 277-288. https://doi.org/10.1159/000064443

Identification of differentially expressed cDNA clones from gerbil cochlear outer hair cells. / Zheng, Jing; Long, Kevin B.; Robison, Donald E.; He, David Z.Z.; Cheng, Jennifer; Dallos, Peter; Madison, Laird D.

In: Audiology and Neuro-Otology, Vol. 7, No. 5, 09.10.2002, p. 277-288.

Research output: Contribution to journalArticle

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T1 - Identification of differentially expressed cDNA clones from gerbil cochlear outer hair cells

AU - Zheng, Jing

AU - Long, Kevin B.

AU - Robison, Donald E.

AU - He, David Z.Z.

AU - Cheng, Jennifer

AU - Dallos, Peter

AU - Madison, Laird D.

PY - 2002/10/9

Y1 - 2002/10/9

N2 - 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.

AB - 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.

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KW - Nonsyndromic deafness loci

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KW - Outer hair cells (OHC)

KW - Prestin

KW - Subtracted library

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EP - 288

JO - Audiology and Neuro-Otology

JF - Audiology and Neuro-Otology

SN - 1420-3030

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