[Construction and expression of adeno-associated virus vectors of Smad 6 and Smad 7 genes in human renal tubule epithelial cells].
OBJECTIVE
To construct the adeno-associated virus(AAV) vectors of Smad 6 and Smad 7 genes and observe their expressions in human renal tubule epithelial cells.


METHODS
The plasmids pcDNA3-Smad 6/flag and pcDNA3-Smad 7/flag were digested with BamH I and Xho I. Then the Smad 6/flag and Smad 7/flag gene fragments were cloned into plasmid pAAV-MCS, respectively to construct the recombinant pAAV-Smad 6/flag and pAAV-Smad 7/flag plasmids. The recombinant expression plasmid or pAAV-LacZ plasmid were co-transfected into the HEK 293 cells with pHelper and pAAV-RC by calcium-phosphate precipitation method. Recombinant AAV-2 viral particles were prepared from infected HEK293 cells and then were used to infect human renal tubule epithelial cells (HKCs), The expressions of Smad 6 and Smad 7 in HKCs were demonstrated by immunocytochemical staining.


RESULTS
The recombinant AAV vectors of Smad 6 or Smad 7 genes were constructed and expressed in the HKCs successfully.


CONCLUSIONS
These results indicate that AAV can deliver Smad 6 and Smad 7 genes to renal cells in-vitro, suggesting the recombinant AAV can be used for gene therapy of renal fibrosis.
Huang
Yun-Jian
YJ
Department of Nephrology, Xinqiao Hospital, Chongqing 400037, China. yun81@sina.com
Zhao
Jing-Hong
JH
Yang
Tang-Jun
TJ
Zhang
Jin-Hai
JH
Cai
Wen-Qin
WQ
D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D000229:Dependovirus / Q000235:genetics / Q000378:metabolism*; D004622:Embryo, Mammalian; D004847:Epithelial Cells / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007668:Kidney / Q000166:cytology; D007684:Kidney Tubules / Q000166:cytology / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D051905:Smad6 Protein; D051906:Smad7 Protein; D015534:Trans-Activators / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; C497802:SMAD6 protein, human; C497806:SMAD7 protein, human; D051905:Smad6 Protein; D051906:Smad7 Protein; D015534:Trans-Activators