I agree with Dr. Mosier, but I'll add my two cents - this applies to retroviral vectors like the Moloney viruses grown in amphotropic packaging lines. I would be careful assuming that human cells infected with these are not producing viruses - they should be tested in a helper assay, so that you know the level at which the uncertainty exists. If the packaging lines are carrying a low level of helper, then these can be transfered and the human cells can produce virus. A few years ago, we produced human tracheal cell lines by culturing explants on irradiated Psi-Crip cells that had been produced by ping-ponging with Psi-CRE. The resulting human cell lines produced significant levels of infectious virus (carrying SV40 T antigen). So, I would have the investigators check since you don't know how they have treated their packaging cells. Bleach should kill the virus without any problem. The viruses are fragile - the half-life at room temperature is about an 1 hour (I believe). They should also be susceptible to detergent and airborne RNases. The other thing to keep in mind is that you probably have to work at getting infected with mouse retroviruses - the target cell has to be cycling, and in vitro, we usually add an agent to reduce the repulsive charges between the virus and the cell. On top of that we have immune systems. That doesn't mean you can't be infected. Over-all these are considered low risk vectors. Here's some references: Introduction to the Biosafety Guidelines for the Sorting of Unfixed Cells. Cytometry 28: 97-98 (1997) Biosafety Guidelines for the sorting of Unfixed Cells. Cytometry 28: 99-117, (1997) ISAC has a safety committe and I believe Ingrid Schmid is Chair <schmid@mednet.ucla.edu> ----------------- James W. Jacobberger Associate Professor of Oncology Case Western Reserve Univ Cleveland, OH 44106 -----Original Message----- From: Donald E. Mosier [mailto:dmosier@scripps.edu] Sent: Wednesday, January 27, 1999 2:04 PM To: cyto-inbox Subject: Re: Retroviral vectors 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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