[Isolation and identification of greater epithelial ridge in rat cochlea].
OBJECTIVE
To investigate the method for isolating greater epithelial ridge by use of thermolysin digestion combined with dissection under microscope.


METHODS
The basement membrane prepared from postnatal day 0 to postnatal day 3 rat was placed into D-Hanks solution with thermolysin and DNase, then incubated at 37 degrees C for 25 minute and dissected under microscope. After GER had been isolated, morphological comparison, immunohistochemistry, RT-PCR and GER culture were performed for the purpose of identification of GER.


RESULTS
Immunohistochemistry indicated that the isolated GER was purely epithelial tissue. RT-PCR analysis certified that the isolated GER expressed Hes1 which was selectively expressed in GER at early postnatal in cochlea. The isolated GER could be successfully cultured in the presence of 5% serum and could survive at least 12 day in the serum-free medium.


CONCLUSIONS
GER isolation can be successfully performed by use of thermolysin combined with microdissection. This method may provide a good technique to establish the GER cell lines or perform series of gene transfection experiments.
Song
Wei
W
Institute of Otolaryngology of Chinese PLA, Beijing, 100853, China. sw629@126.com
Hu
Yinyan
Y
Guo
Wei
W
Zheng
Guiliang
G
Zhai
Suoqiang
S
Gao
Weiqiang
W
D000818:Animals; D001485:Basement Membrane / Q000166:cytology*; D002454:Cell Differentiation; D002478:Cells, Cultured; D003051:Cochlea / Q000166:cytology*; D004848:Epithelium; D007552:Isotonic Solutions; D042282:Microdissection; D009924:Organ Culture Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley
C586711:D-Hanks' solution; D007552:Isotonic Solutions