[Construction of GJB2 mutations common in Chinese EGFP fusion protein vectors].
OBJECTIVE
To construct GJB2 gene mutations common in Chinese EGFP fusion protein vectors, and to search for better way to study the mechanism of deletion mutations in GJB2 gene.


METHODS
Non-fusion protein vectors of 235delC, 299-300 del AT and 176 del 16 bp were first made by point mutation methods in vitro. Then expression part of the upper 3 mutations were amplified by PCR and the PCR products were cloned into TA cloning vector. After cutting by restriction enzymes EcoRI/BamHI, three deletion mutations were inserted into pEGFP-N1 vector. Sequencing was used to verify the validity of the fusion protein vectors. HEK293 cells were transfected with the recombinant DNA samples by the liposome complex method.


RESULTS
The recombined plasmids were highly expressed in HEK293 cells. Green fluorescence signals were distributed uniformly in cytoplasm.


CONCLUSIONS
GJB2 mutations common in Chinese EGFP fusion protein vectors were constructed successfully. It may provide a better way to explore the reasons of nonsyndromic hearing loss common in Chinese.
Zhang
Yanping
Y
Department of Otolaryngology, the Second Affiliated Hospital of General Hospital of Chinese PLA, Beijing, 100091, China. yzhan28@163.com
Zhang
Yuanding
Y
Li
Lina
L
Ma
Lei
L
Sun
Yurui
Y
Zhang
Zonglin
Z
Liu
Jinwei
J
Deng
Huiyan
H
Zhu
Wei
W
D044466:Asian People / Q000235:genetics; D000072259:Connexin 26; D017630:Connexins / Q000235:genetics*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D017384:Sequence Deletion
D017630:Connexins; C000607042:GJB2 protein, human; C485184:enhanced green fluorescent protein; D000072259:Connexin 26; D049452:Green Fluorescent Proteins