3 
the strain (s) of E. coll ; the topology and orientation of the inserted 
DNA (chiefly, whether present as a tandem insertion or a single copy); 
the route of administration; whether colonization occurs, the 
presence or absence of intestinal bacteria, the nature of the Intestinal 
flora, and the age of the mice when first exposed. 
Carrying out these experiments will also provide an opportunity to 
study the related question of the possible biohazard of handling bio- 
logically active DNA, and we plan to include studies of the infectivity 
of polyoma DNA administered by various routes. 
At the present time we foresee these studies being carried out in 
three stages. Depending on the results obtained and barring any un- 
anticipated technical problems, stages 1 and 2 should be completed within 
6 to 8 months. The third stage, which will Involve the use of a plasmid 
vector system, will require the cloning and selection of bacteria con- 
taining the desired recombinant molecules under P4 conditions. We 
anticipate that stage three can be completed within nine months to a 
year. 
The experiments enumerated below represent our current ideas re- 
garding how such an endeavor should proceed. In no way should they be 
construed as the final protocol for the studies proposed. We are presently 
contacting investigators both within and outside the NIH who we feel can 
make a significant contribution toward the planning and execution of the 
project. 
STAGE 1. Infection of Mice with Biologically Active and Potentially 
Biologically Active Materials 
This stage will have two objectives: to determine the infectivity 
of polyoma DNA when given by ingestion, inhalation and injection; and to 
provide certain baseline data needed for design of the mouse experiments. 
[Appendix C — 80] 
