The nonviral episomal replicating vector pEPI-1 allows long-term inhibition of bcr-abl expression by shRNA.
The inhibition of gene expression by RNA interference harbors a high potential for application in the therapy of human diseases. However, while exogenous application of siRNAs efficiently inhibits gene expression, these effects are only transient in mammalian cells. We designed a short hairpin RNA-expression cassette to target the bcr-abl oncogene that was then introduced into the nonviral vector system pEPI-1, which replicates episomally in the absence of selection in the bcr-abl-positive cell line K562. Forty-two days after transfection the bcr-abl- but not the cytokine-dependent growth rate was found to be drastically reduced in K562 cells. Western analysis revealed a more than 90% reduction in the expression of the fusion protein bcr-abl while the expression of the bcr protein remained unaffected. In addition, we show that the level of bcr-abl mRNA was specifically reduced in these cells for more than 90%. These results demonstrate that the vector system pEPI-1 allows specific and efficient long term gene suppression by using a short hairpin RNA transcription unit.
Jenke
Andreas C W
AC
Children's Hospital, Helios Klinikum Wuppertal, 42283 Wuppertal, Germany.
Eisenberger
Tobias
T
Baiker
Armin
A
Stehle
Isa M
IM
Wirth
Stefan
S
Lipps
Hans Joachim
HJ
D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D049109:Cell Proliferation; D016207:Cytokines / Q000378:metabolism; D004258:DNA Polymerase III / Q000235:genetics; D016044:Fusion Proteins, bcr-abl / Q000187:drug effects / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics* / Q000494:pharmacology; D006801:Humans; D020014:K562 Cells; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D016337:RNA, Catalytic / Q000737:chemistry; D014779:Virus Replication / Q000235:genetics*
D016207:Cytokines; D016337:RNA, Catalytic; C418481:hairpin ribozyme; D012313:RNA; D016044:Fusion Proteins, bcr-abl; D004258:DNA Polymerase III