Stable integration of a functional shRNA expression cassette into the murine leukemia virus genome

Short hairpin RNA (shRNA) can be stably expressed in cells to down-modulate gene expression. While retroviral and lentiviral vectors can be used to deliver shRNAs, the restricted viral titer is the major limitation for efficient gene transfer, which is especially important for cancer gene therapy. We were interested in using replicating murine leukemia virus (MLV) to enhance the shRNA transfer. Although stem loop structures could potentially interfere with the retroviral life cycle, we were able to demonstrate that the insertion of shRNA expression cassettes into MLV did not interfere significantly with viral fitness. The virus was genetically stable and able to silence target gene expression. Our results show that replicating MLVs are excellent tools for delivering shRNAs efficiently throughout the culture and have the potential to be used for gene function elucidation or even for cancer gene therapy.