MOLECULAR DISSECTION OF THE ORGAN OF CORTI

Project: Research project

Description

DESCRIPTION (provided by applicant): The mouse inner ear offers an excellent
paradigm to characterize and analyze the functional genomics of unique and rare
cell types. We propose to utilize the specialized cells of the organ of Corti
(OC) for construction and analysis of full-length, microquantity cDNA (mq-cDNA)
libraries and the development of an OC UNIGENE set for expression analyses. We
wifi utilize our current mq-cDNA protocol with a number of cell procurement
strategies to construct representative immortalized full-length cDNA libraries
from the cells of the OC with a focus on the supporting cells. In specific aim
1, cochlear hair cells (HCs) provide relatively "known" cell types for
assurance of the quality of both the resulting "immortalized" mq-cDNA templates
and subsequent mqcDNA libraries. For expression profiling and cell procurement
we will test the value of utilizing a Chnra9-GFP transgenic mouse line that
express green fluorescent protein in cochlear hair cells. In specffic aim 2,
subtracted normalized cell-specific cDNA libraries will be constructed which
represent the supporting cells within the OC. These are inner phalangeal,
border cells of the inner sulcus, inner pifiar, outer pifiar, Deiters?, and
Hensen?s cells, as well as the bordering Claudius? cells. Cell procurement
protocols, e.g., microdissection, cell plucking, and if needed laser-capture
microscopy, will be used with the mq-cDNA protocol for full-length cDNA library
production. GFP-expressing transgenic mice will be used to assist in isolation
of specffic types of OC supporting cell. Quality assessment of these cDNAs will
be accomplished by using in silico microarray analyses to detect expression of
ion channel genes, rare to common housekeeping genes, developmentally expressed
genes, cell-specific genes of the OC, and genes expressed in only
non-sensory/non-neuronal cells. In specific aim 3 the cell-specific clones
derived from aims 1 & 2 will be used to generate a microarray chip containing
an OC "UNIGENE" set. Further identification of novel and/or new transcripts
wifi be done by the combined use of suppressive subtractive hybridization with
microarray analyses. Specific aim 4 will use differential expression analysis
to verify the cellular specificity of these clones using the OC chip for in
silico microarray analyses in combination with null and spontaneous mutant
mice. Extrapolation of the expression profiles may provide insights into the
functional characteristics and status of each of these cell types in normal and
pathogenic states of the OC.
StatusFinished
Effective start/end date7/17/026/30/08

Funding

  • National Institutes of Health: $351,732.00
  • National Institutes of Health: $142,454.00
  • National Institutes of Health: $353,864.00
  • National Institutes of Health: $341,402.00
  • National Institutes of Health: $80,360.00
  • National Institutes of Health: $331,473.00
  • National Institutes of Health: $193,887.00

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Organ of Corti
Dissection
Complementary DNA
Auditory Hair Cells
Microarray Analysis
Gene Library
Transgenic Mice
Genes
Microdissection
Essential Genes