[Construction of recombinant adenovirus including microdystrophin and expression in the mesenchymal cells of mdx mice].
Construction of recombinant adenovirus, which contain human microdystrophin, and then transfection into mesenchymal cells( MSCs) of mdx mice were done, and genetically-corrected isogenic MSCs were acquired; the MSCs transplantation into the mdx mice was then done to treat the Duchenne muscular dystrophy( DMD). Microdystrophin cDNA was obtained from recombinant plasmid pBSK-MICRO digested with restrictive endonuclease Not I ; the production was inserted directionally into pShuttle-CMV. The plasmid of pShuttle-CMV-MICRO was digested by Pme I , the fragment containing microdystrophin was reclaimed and transfected into E. coli BJ5183 with plasmid pAdeasy-1. After screening by selected media, the extracted plasmid of positive bacteria was transfected into HEK293 cells with liposome and was identified by observing the CPE of cells and by the PCR method. Finally, MSCs of mdx mice were infected with the culture media containing recombinant adenovirus, and the expression of microdystrophin was detected by RT-PCR and immunocytochemistry. Recombinant adenovirus including microdystrophin was constructed successfully and the titer of recombinant adenovirus was about 5.58 x 10(12) vp/mL. The recombinant adenovirus could infect MSC of mdx mice and microdystrophin could be expressed in the MSC of mdx mice. Recombinant adenovirus including microdystrophin was constructed successfully, and the microdystrophin was expressed in the MSC of mdx mice. This lays the foundation for the further study of microdystrophin as a target gene to correct the dystrophin-defected MSC for stem cell transplantation to cure DMD.
Xiong
Fu
F
Center for Stem Cell Biology and Tissue Engineering of Sun Yat-sen University Guangzhou 510080, China.
Zhang
Cheng
C
Xiao
Shao-Bo
SB
Li
Mei-Shan
MS
Wang
Shu-Hui
SH
Yu
Mei-Juan
MJ
Shang
Yan-Chang
YC
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002478:Cells, Cultured; D016189:Dystrophin / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D045164:Mesenchymal Stem Cell Transplantation / Q000379:methods; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D018101:Mice, Inbred mdx; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics / Q000473:pathology / Q000628:therapy; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014161:Transduction, Genetic
D016189:Dystrophin; D011993:Recombinant Fusion Proteins