Suicide genes for negative selection of cells have been powerful tools in somatic cell genetic studies and in gene therapy. Here we report on the construction, characterization, and utilization of retroviral vectors encoding barnase, a ribonuclease from Bacillus amyloliquefaciens, expression of which results in apoptosis of transduced mammalian cells. High-titer viral vector production was enabled by expression of an inhibitor of barnase (barstar) in transfected cells generating murine leukemia virus (MLV)- and HIV-1-based vectors. To identify cellular genes required for infection we used barnase-encoding vectors in a genetic screen to isolate mutant mammalian cells that are resistant to infection by MLV and HIV-1. We describe one such mutant clone that is inhibited in the infection process after reverse transcription. These results suggest that barnase-encoding vectors should be useful for negative selection strategies examining retroviral infection from entry to integration. Furthermore these vectors could have utility in approaches for gene therapy that require specific cell ablation.
Bibliographical noteFunding Information:
We thank Scott McIvor and Kathleen Conklin for critical reading of the manuscript and helpful suggestions. This work was supported by startup funds from the Institute of Human Genetics, University of Minnesota and the University of Minnesota Medical School, and a NIH grant (R21 AI60470). S.A. was a recipient of a student fellowship from the Institute of Human Genetics, University of Minnesota.
- cell mutants
- negative selection
- retroviral vectors
- suicide vectors