Letter from Herbert W. Boyer, Philip Coffino, and Harold Varmus to Daphne Kamely, National Institute of General Medical Sciences
To ensure the safety of recombinant DNA research (research that, among many other results, yielded important techniques for
Varmus' investigation into the origins of cancer), the National Institutes of Health in 1976 issued guidelines for the
construction and operation of laboratories in which such research was conducted. The guidelines established four containment
levels, setting more stringent requirement for such features as air exhaust and wastewater disposal systems with each ascending
level. This directive lays out the protocol for using and cleaning a level two containment facility in Varmus and Bishop's
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1977-05-26 (May 26, 1977)
Boyer, Herbert W.
National Institute of General Medical Sciences
Original Repository: University of California, San Francisco. Archives and Special Collections. Harold E. Varmus Papers
Reproduced with permission of the Regents of the University of California.
We would like to undertake a project involving recombinant DNA. Under the present NIH guidelines, this project would appear
to require P4 containment. We request that the Recombinant DNA Advisory Committee review the following considerations which
we feel would warrant P3 containment conditions.
We propose to use polyoma virus as a vector to clone the thymidine kinase (TK) gene of pseudorabies virus (PRV) in mouse cells.
DNA of a polyoma mutant temperature sensitive in an early function (ts-a) will be purified. Restriction endonuclease(s) will
be used to delete a portion of the genome that codes for late function. A PRV restriction fragment that codes for TK will
be identified and purified using transfection of TK-mouse L cells to HAT resistance as an assay of activity. Recombinants
of the polyoma and TK fragments will be formed using conventional procedures. TK-L cells will be infected at the restrictive
temperature and cells selected in HAT medium to obtain clones that express TK activity. These cells will be shifted to the
permissive temperature and closed circular DNA isolated from the Hirt supernatant.
The following considerations apply in evaluating the biohazards of the proposed experiment:
1. PRV, a herpes virus, is not known to be pathogenic for man, but is pathogenic for pigs and cattle.
2. Viral strains with highly attenuated virulence for livestock and laboratory animals have been described and will be used
3. Polyoma with a deletion of late function will be used.
4. No virions will be generated.
5. A partially defined fragment of PRV DNA will be used.
6. Mouse cells are permissive for both polyoma and PRV, hence both genomes have had the opportunity to coexist in a single
host before the introduction of recombinant DNA technoloqy.
In view of these considerations, we request permission to conduct this series of experiments under P3 containment conditions.
This project will be conducted by the undersigned.