Construction of adenovirus for high level expression of small RNAs in mammalian cells. Application to a Bcl-2 ribozyme.
A series of plasmid vectors have been generated to allow the rapid construction of adenoviral vectors designed to express small RNA sequences. A truncated human U6 gene containing convenient restriction sites has been shown to be expressed at high levels following electroporation into a series of human cell lines. This gene was ligated into a promoterless adenoviral plasmid, and we have generated high titer virus by homologous recombination with adenoviral Addl327 DNA in 293 cells. Recombinant adenovirus containing a hammerhead ribozyme sequence targeted toward the Bcl-2 mRNA has been used to transduce a panel of human tumor cell lines. We have demonstrated high level expression of the recombinant U6 gene containing the ribozyme and reduction of Bcl-2 protein in transduced cells. These plasmids are suitable for the development of adenoviral vectors designed to express both ribozymes and antisense RNA in human cells.
Potter
P M
PM
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
McKenzie
P P
PP
Hussain
N
N
Noonberg
S
S
Morton
C L
CL
Harris
L C
LC
D000256:Adenoviridae / Q000235:genetics*; D015153:Blotting, Western; D017931:DNA Primers / Q000737:chemistry; D004279:DNA, Viral / Q000235:genetics*; D015870:Gene Expression; D018390:Gene Targeting; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids; D016133:Polymerase Chain Reaction; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics* / Q000378:metabolism; D016372:RNA, Antisense / Q000494:pharmacology; D016337:RNA, Catalytic / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000378:metabolism
D017931:DNA Primers; D004279:DNA, Viral; D019253:Proto-Oncogene Proteins c-bcl-2; D016372:RNA, Antisense; D016337:RNA, Catalytic; D012333:RNA, Messenger; D017414:Ribonucleoprotein, U4-U6 Small Nuclear