[Transfection and in vitro expression of human microdystrophin gene in rat mesenchymal stem cells].
OBJECTIVE
To construct the eukaryotic expression vector of human microdystrophin gene and observe its expression in rat mesenchymal stem cells (rMSCs) in vitro.


METHODS
The plasmid PBSK-MICRO containing human microdystrophin cDNA was digested by restriction endonuclease, and the resultant microdystrophin fragment was inserted into the NotI site of pcDNA3.1(+) to prepare the eukaryotic expression vector-pcDNA3.1(+)/ microdystrophin, which was identified by endonuclease digestion and sequencing. The recombinant plasmid was transfected into rMSCs via lipofectamine, and after G418 selection, the expression of microdystrophin was detected by RT-PCR and indirect immunofluorescence assay.


RESULTS
Microdystrophin gene fragment was correctly inserted into the plasmid pcDNA3.1(+), as conformed by sequencing and digestion with Not I and Hind III. The total mRNA of the transfected rMSCs was extracted and microdystrophin mRNA expression was found in the cells by RT-PCR. Indirect immunofluorescence assay for the protein expression of microdystrophin showed bright red fluorescence in the transfected rMSCs.


CONCLUSIONS
Eukaryotic expression plasmid pcDNA3.1(+)/microdystrophin has been constructed successfully and microdystrophin can be expressed in transfected rMSCs in vitro, which may facilitate further research of Duchenne muscular dystrophy treatment by genetically modified allogeneic stem cell transplantation.
Wang
Shu-hui
SH
Department of Neurology, First Affiliated Hospital1, Sun Yat-sen University, Guangzhou 510080, China. wangshuhui2008@163.com
Zhang
Cheng
C
Chen
Song-lin
SL
Yu
Mei-juan
MJ
Zhang
Ya-ni
YN
Li
Mei-shan
MS
Xiong
Fu
F
Shang
Yan-chang
YC
Feng
Shan-wei
SW
Shen
Ben-chang
BC
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D016189:Dystrophin / Q000096:biosynthesis / Q000235:genetics*; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D016189:Dystrophin; D010446:Peptide Fragments; D012333:RNA, Messenger