Construction and use of retroviral vectors encoding the toxic gene barnase.

Abstract

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.

• Authors: Sumit Agarwal, Bryan Nikolai, Tomoyuki Yamaguchi, Patrycja Lech, Nikunj V Somia
• Journal: Molecular therapy : the journal of the American Society of Gene Therapy (Mol Ther) Vol. 14 Issue 4 Pg. 555-63 (Oct 2006) ISSN: 1525-0016 [Print] United States
• PMID: 16814610 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• Bacterial Proteins
• Ribonucleases
• Bacillus amyloliquefaciens ribonuclease

Topics

• Animals
• Bacterial Proteins
• Cell Line
• Cell Proliferation
• Cell Separation
• Cricetinae
• Genetic Vectors (genetics)
• Humans
• Lentivirus (genetics)
• Leukemia Virus, Murine (genetics)
• Male
• Mutation (genetics)
• Ribonucleases (biosynthesis, genetics, toxicity)
• Transcription, Genetic (genetics)