[Adenovirus-delivered BMI-1 shRNA].
Recently, some plasmid vectors that direct transcription of small hairpin RNAs have been developed, which are processed into functional siRNAs by cellular enzymes. Although these vectors possess certain advantages over synthesized siRNA, many disadvantages exist, including low and variable transfection efficiency. This study was aimed to establish an adenoviral siRNA delivery system without above-mentioned disadvantages on the basis of commercially available vectors. A vector was designed to target the human polycomb gene BMI-1. The pAd-BMI-1shRNA-CMV-GFP vector was produced by cloning a 300 bp U6-BMI-1 cassette from the pGE1BMI-1shRNA plasmid and a CMV-GFP cassette from pAdTrack CMV in pShutter vector. The adenovirus was produced from the 293A packaging cell line and then infected K562 cells. The mRNA and protein levels of Bmi-1 were detected by real time-PCR and Western blot respectively. The results showed that the adenovirus carrying the BMI-1shRNA was successfully produced. After being transfected with the adenovirus, the K562 cells dramatically down-regulated BMI-1 expression, whereas the adenoviruses carrying control shRNA had no effect on BMI-1 expression. It is concluded that the adenoviruses are efficient vectors for delivery of siRNA into mammalian cells and may become a candidate vector carrying siRNA drugs for gene therapy.
Hematology Center, Beijing Children Hospital Affiliated to Capital University of Medical Sciences, Beijing 100045, China. chenzhp@yahoo.com.cn
Zhen-Ping
ZP
Chen
Xiao-Li
XL
Chen
Jie
J
Zhen
D000256:Adenoviridae / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D020014:K562 Cells; D009687:Nuclear Proteins / Q000235:genetics*; D010957:Plasmids; D063150:Polycomb Repressive Complex 1; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics*; D014162:Transfection
C069077:BMI1 protein, human; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D063150:Polycomb Repressive Complex 1