1MUA-18 
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1 1 . EXPERIME NTAL RATIONALE 
During the past two years, considerable interest has developed regarding the 
potential benefits and biohazards associated with the production of recombinant 
DNA molecules and their subsequent amplification in both prokaryotic and 
eukaryotic cells. 
Many individuals have felt that an experiment involving the cloning of polyoma 
virus DNA in Escherichia coli , using either a lambdaphage or plasmid vector, 
and the subsequent inoculation of mice with the resultant organisms would be 
highly informative. We have been charged by the NIH Recombinant DNA Molecule 
Program Advisory Committee to plan and carry out such experiments utilizing 
facilities of suitable containment. 
The reasons for selecting polyoma virus for such an experiment are manifold. 
Polyoma virus DNA is small (3 x 10^ daltons), is well characterized chemically 
and physically, and is infectious in mice. The virus does not appear to 
replicate in human cells (Eddy, B. E. , Virol. Monogr. , 7_: 114, 1969; Pollack, 
R. E. et_ al_. , J. Cell Physiol., 77_: 117, 1971) presumably because it fails to 
adsorb or be uncoated. Microinjection of polyoma virions or supercoiled DNA 
into the cytoplasm of human cells in culture results ir. the synthesis of viral 
specific T antigen; no viral capsid (V) antigen or infectious virus was 
detected following such manipulation (Gruen ert aj_. , Virology !58: 290, 1974). 
However, the chief reason for its selection .is the wel i -documented observation 
that parenteral inoculation of mice with minute doses of virus reproducibly 
leads to an immune response (Rowe et_ a]_. , Journal of Experimental Medicine 
1 09 : 379,' 1959; Rowe ert al_. , Perspectives in Virology 2_: 177, 1961 ). 
Feeding and intranasal instillation of virus are much less efficient modes 
of infection. This phenomenon is the basis for the mouse antibody production 
(MAP) test for polyoma virus. In this assay, mice from a polyoma-free colony 
are injected with test material and, three to four weeks later, are tested 
for serum hemagglutination inhibiting (HI) antibody against the virus. This 
test can detect approximately one tissue culture infectious dose (TCID) of 
polyoma virus: adults are as sensitive as suckling mice. We thus view the 
HI antibody to be a highly sensitive, unambiguous and convenient test 
reflecting the occurrence of a productive polyoma infection in mice. It is 
especially appealing in the context of the proposed studies which involve the 
use of an £. col i cloning system because it is a continuing test ( i . e . , a 
single group of mice, colonized by an E. col i plasmid or phage system 
containing a polyoma component, can be tested repeatedly for whether produc- 
tive virus infection occurs during a prolonged perioo of observation). A 
second assay that will be employed to evaluate the oncogenic potential of 
bacteria containing a polyoma-DNA recombinant involves the i ntraperi toneal 
inoculation of test materials into newborn hamsters. In this assay (Stoker, 
British Journal of Cancer, J4_: 679, 1960) polyoma vi rus- i nduced tumors 
involving the kidneys, heart, peritoneum, liver and lungs appear 2 to 4 weeks 
following injection: the number of foci of tumor cells in the kidneys can be 
roughly correlated with the initial virus dose. In tnis test system only a 
portion of the viral genome must function (viz. the "early" gene sequences 
containing the genetic in-formation for "oncogenicity") for a positive result. 
This assay may prove most useful in this risk-evaluation study, particularly 
[Appendix C — 232] 
