[Construction of eukaryotic expression vector of congenital long QT syndrome related HERG gene A561V mutation and its expression in HEK293 cells].
OBJECTIVE
To investigate the protocol of the construction of HERG gene mutations, an A561V mutation which was detected in a Chinese congenital long QT syndrome (LQTS) family had been constructed and expressed in vitro.


METHODS
The A561V cloning vector PGEM-HERG-A561V was constructed by quick site-directed mutagenesis PCR. The A561V expressive vector pcDNA3-HERG-A561V was constructed by restriction enzymes. pRK5-GFP was cotransfected with pcDNA3-HERG-A561V or wild type pcDNA3-HERG into HEK293 cells by Superfect transfection reagent. The protein was measured by immunofluorescence.


RESULTS
Direct sequence analyses revealed a C to T transition at position 1682. The A561V mutation was correctly combined to eukaryotic expressive vector pcDNA3 and expressed in HEK293 cells. The protein of mutation was expressed in cytoplasm and cellular membrane while the wild type gene was expressed only on cellular membrane.


CONCLUSIONS
The protocol can be used successfully to construct and express HERG A561V mutation and it forms the basement of the further study on functions of mutation.
Li
Yu
Y
Department of Cardiology, First Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, 710061, P. R. China.
Cui
Chang-cong
CC
Huang
Chen
C
Lian
Jiang-fang
JF
Zhao
Yong-hui
YH
Xue
Xiao-lin
XL
Zhao
Xiao-ge
XG
D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004252:DNA Mutational Analysis; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008133:Long QT Syndrome / Q000235:genetics*; D008856:Microscopy, Fluorescence; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004247:DNA