The current retrovirus vectors used for gene expression in human cells are based either on mouse retroviruses (typically Moloney mouse leukemia virus) or on primate lentiviruses including HIV. All such systems use defective virus genomes that are packaged into infectious particles that are only capable of a single round of replication. The only defined safety concern is that these defective virus genomes might potentially be rescued by what is called a helper virus if that replication competent virus were to infect the same cell. This has happened in early monkey trials of mouse retrovirus-mediated gene transfer, and the helper virus was an endogenous monkey retrovirus. Since no replication competent endogenous human retroviruses are known, the risk of this happening in humans is thought to be remote. There are human endogenous retrovirus sequences (HERV) dispersed through the human genome, but none of these encodes a complete virus sequence. These is a very remote possibility that activation of one of these HERV sequences could complement what is missing in the original retrovirus vector (typically the envelope gene). In short, the biohazard posed by retrovirus-transduced human cells is very, very low but potentially not zero. It is much more important to know if the human cells harbor HIV or hepatitis B or C in terms of biohazard risk. Our laboratory handles such cells under BSL-2 conditions, and flow cytometry is performed on fixed samples (1% paraformaldehyde for 30 min.). Donald Mosier >Dear Flow Group, > >Could someone please bring me up to speed on sorting human cells infected >with retrovirus which are used for research purposes? I understand that >these reagents are supposed to be defective viruses used for genetic >transfer. What biohazard classification has a laboratory working with these >vectors? What precautions over and above the common practices are required? >Will bleaching through the sample probe between sorts eliminate viruses from >the flow cytometer to prevent viral contamination of subsequent sorts? > >Please cite references if available. Can someone cite a reference that >lists the requirements for laboratories according to the different biohazard >classifications? > >Thanks in advance, >Sue Rice > ______________________________________________________________________________ Donald E. Mosier, Ph.D., M.D. Department of Immunology IMM7 The Scripps Research Institute 10550 N. Torrey Pines Road La Jolla, CA 92037 619 784-9121 voice 619 784-9190 fax
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