Construction of Adenovirus for High Level Expression of Small RNAs in Mammalian Cells Application to a Bal-2 Ribozyme

摘要

A series of plasmid vectors have been generated to allow the rapid construction of adenoviral vectors designed to express small RNA seqences. A truncated human U6 gene containing conventient 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 Add1327 DNA in 293 cells. Recombinant adenivirus 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 riboozyme 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.