A minidystrophin-EGFP fusion gene expressed in Cos-7 cells mediated by human source vector.
OBJECTIVE
To construct a human source vector containing minidystrophin-EGFP fusion gene and investigate its expression in Cos-7 cells.


METHODS
The recombinant human source vector named pHrnDysG was constructed with PCR-clone methods. Three fragments of dystrophin gene were PCR amplified from normal human dystrophin gene cDNA (GenBank NM04006). These three fragments were ligated to generate a minidystrophin gene. The enhanced green fluorescent protein (EGFP) gene was fused to the C terminal of the minidystrophin gene, and then the pHrnDysG was finally obtained by cloning the fusion gene to pHrneo. Fluorescence microscope and RT-PCR were used to detect the expression of minidystrophin-EGFP fusion gene after the recombinant construct was transfected into Cos-7 cells by lipofectamine.


RESULTS
Restrictive enzyme digestion analysis and sequencing confirmed that pHrnDysG vector was constructed successfully. After the recombinant pHrnDysG was transfected to Cos-7 cells, RT-PCR demonstrated that the fusion gene was successfully transcribed, and the green fluorescence was observed at the cell membrane.


CONCLUSIONS
The minidystrophin-EGFP fusion gene mediated by pHrneo vector could express in Cos-7 cells and its products' localization in the cell membrane was the same as that of full length dystrophin. These results suggested that the recombinant human source vector pHrnDysG might be potentially used in studies on the gene therapy of Duchenne muscular dystrophy.
Liang
Yu
Y
National Laboratory of Medical Genetics, Central South University, Changsha, Hunan, PR China.
Liang
De-sheng
DS
Xue
Zhi-gang
ZG
Long
Zhi-gao
ZG
Wu
Ling-qian
LQ
Pan
Qian
Q
Hu
Yi-qiao
YQ
Dai
He-ping
HP
Xia
Kun
K
Xia
Jia-hui
JH
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D016189:Dystrophin / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D016189:Dystrophin; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins