desired plasmid DNA will be grown in mass culture in the presence of 
chloramphenicol to amplify the plasmid element. 
5. Plasmid DNA will be purified from infected cultures and 
assessed for the presence of polyoma DNA sequences with appropriate 
restriction enzymes and heteroduplex techniques. 
C. Biological Activity of Cloned Becoinbinant DNA 
(Carried out under P- A condi tions ) . 
1. Infection of mouse cells in culture 
Primary mouse embryo cells in culture will be infected 
with purified recombinant plasmid DNA in the presence of DEAE-dextran. 
Polyoma virus will be detected by CPE, indirect immunofluorescence of 
the infected cells and a blind passage thereof. 
2. Infection of mice 
a) Adult and newborn mice will be inoculated subcutaneousl 
and intraperitoneally with varying amounts of E. coli containing Col El- 
polyoma recombinant plasmids, a bacterial cell lysate, and with purified 
recombinant plasmid DNA. 
b) If polyoma HI antibody develops in the animals inoculate 
parenterally , germ-free and conventional mice will be infected with 
varying amounts of J5. coll containing recombinant plasmids by the oral 
and nasal routes. 
c) If polyoma HI antibody is not detected following the 
parenteral administration of recombinant plasmid and plasmid DNA, mice 
will be inoculated with a bacterial clone containing a different plasmid 
DNA recombinant by the parenteral route. The recombinant plasmid from 
the initial clone would not be used to infect mice by the oral and nasal 
routes since it sppids very unlikely that seroconversion would occur in 
the face of a negative result following parenteral administration of 
such plasmid material. 
[Appendix C — 85] 
