Background:
Retroviral expression vectors will be used to transduce genes, including oncogenes, into cells in culture. The vectors may have a variable host-range (ecotrophic, amphotrophic, pseudotyped to increase tropism) but are constructed in such a way that they are replication-defective; in other words they will not produce new viral particles in the infected cells[1]. However, there is always the risk that recombination between packaging plasmids and proviral plasmids will rarely give rise to replication competent vectors and hence the need for special caution. The following procedures are implemented to prevent exposure by laboratory workers and support staff.
Procedures:
1) All work will be carried out in the designated cell culture room (Skirball Institute, third floor, lab 12). Adherence to BSL2 biohazard precautions will be strictly enforced (no eating, smoking or storage of food in the lab). Warnings will be posted in the areas involved in retroviral work.
2) Workers must wear gloves.
3) Cultured cells will be maintained in a designated incubator that will be so labeled. Work with the cells will be carried out in a designated laminar flow hood (Baker SG400, that provides bio-protection at the P2 level at least).
4) No sharps will be used.
5) Contaminated plasticware (tissue culture plates, pipettes, tips etc.) will be inactivated with bleach and disposed in a designated biohazard waste basket (“red bags) that will be eventually autoclaved.
6) Contaminated work surfaces will be sterilized by bleach. Containers of contaminated liquids will be treated with bleach.
7) The U.V. light will be left on for 10 minutes after work in the hood to ensure inactivation of any virus contaminating work surfaces in the hood.
8) Centrifugation of cultures and supernatants will be done only in capped tubes or sealed ultracentrifugation containers.
Reference:
1. Muller, A.J., J.C. Young, et al. (1991) BCR first exon sequences specifically activate the BCR/ABL tyrosine kinase oncogene of Philadelphia chromosome-positive human leukemias. Molecular and Cellular Biology, 11: p. 1785-1792.

Posting on incubators, hoods etc:

ONCOGENIC RETROVIRUSES!!

This incubator has been set aside for work with replication defective amphotrophic retroviruses. These include viruses whose inserts encode oncogenes that can transform cells in culture and hence should be considered potentially oncogenic.
All work with cells containing such viruses should be done in the laminar flow hood. No needles or sharp objects may be used. Worker must wear gloves and wash hands after completing the work. The hood must be decontaminated by turning on the U.V. light for 15 minutes. Contaminated solutions must be treated with bleach or autoclaved prior to disposal. Samples may be spun in centrifuge only when capped.

Updated, September 18, 2004