V.' ASSESSMENT OF THt 
iu Jl\ 
i MUA- 1 6 
-5- 
The experiments described in Section IV fall into at least two different 
categories (cloning of animal viral DNA in an E. col i host-vector system and 
the use of a polyoma vector system in mouse cells) of risk as outlined in 
the NIH Guidelines for Recombinant DNA Research. 
The Guidelines state that P4 + EK2 or P3 + EK3 must be used to isolate DNA 
recombinants that include all or part of an animal virus genome. Since 
certified EK3 vectors are not presently available, tht experiments we have 
outlined will be conducted under P4 containment with approved EK2 host-vector 
systems. These conditions can be met with the P4 Mobile Containment 
Laboratory, NIH, and Building 550, FCRC, and the two approved EK2 host-vector 
systems outlined in Section III. Because of the scope and complexities of 
the studies we plan to carry out, we have compiled a list of various 
activities and laboratory manipulations and grouped them according to the 
level of physical containment under which each will be conducted. 
A. P4 Physical Containment 
1. Propagation of P-lambda DNA Recombinants 
a. Transfection of JE. coli with P-lambda DNA 
b. Identification of plaques which contain P-lambda DNA recombinants 
by colony hybridization 
c. Preparation and purification of P-lambda phage stocks 
d. Preparation of P-lambda infected £. col i 
2. Propagation of P-plasmid DNA Recombinants 
a. Transformation of £. col i with P-plasmid DNA 
b. Identification and isolation of E_. coli c~!ones which contain 
P-plasmid DNA recombinants 
c. Preparation of jE. col i cultures containing P-plasmid DNA 
3. Inoculation of animals wi th* bacteria containing P-lambda or P-plasmid 
DNA, with P-lambda phage stocks, or with purified P-lambda or 
P-plasmid DNAs. 
4. Assay of E. col i lysates containing polyoma-.Tphage for the presence 
of infectious polyoma virus. 
a. Preparation of bacterial lysates suitable for use in tissue 
culture systems 
b. Infection of mouse cell tissue cultures with lysates 
c. Purification rf progeny polyoma virus 
[Appendix C — 235] 
