59 
Similarly, when the initial recombination involves a purified segment 
of the foreign chromosomal DNA, and is then not a shotgun experiment, 
the potential for growth of a hazardous organism will be less, because 
the likelihood is lower that an unsuspected hazardous gene is present 
and because the number of clones that must be examined to obtain 
the desired clone is markedly reduced. The Guidelines define "purified" 
(or "enriched") as meaning that the desired segment represents at 
least 99 percent of the total DNA in the preparation, by weight; and 
further, they require evidence that no harmful genes are present. 
Under such circumstances the investigator may lower the containment 
conditions from these recommended for shotgun experiments with DNA 
of the same source, either by one step in physical containment or by 
one step in biological containment. Thus, for example, shotgun exper- 
iments with DNA from birds require P3 and EK2. A DNA fragment 
from birds that is free from harmful genes, and purified to 99 percent 
purity prior to joining to a vector, would require either P2 and EK2 
or P3 and EKl . 
Eukaryote host -vector systems using defective viral vectors are 
also described. Many recombinant DNA experiments will involve the 
use of systems in which the host cells are eukaryote cells grown singly 
in tissue culture. These host cells are fragile and fastidious, and there 
is little or no chance that a living cell could escape from a laboratory 
in the way that an E. coli cell might. Therefore, containment con- 
siderations focus on the vectors in these experiments. Useful vectors 
may include extrachromosomal DNA elements such as organelle DNA, 
or the DNA of viruses that infect the particular cells of interest. Given 
the current state of technology, viral DNAs are most likely to be used 
as vectors in the near future. Animal viruses can escape a laboratory 
in a viable form, especially if laboratory workers become infected with 
them. 
Two animal viruses are technically feasible for use at present; 
they are called "SV40" and "polyoma. ' Both SV40 and polyoma are 
oncogenic --that is, they cause tumor formation in newborn small 
laboratory mammals— and both can transform a variety of cells of 
mammalian origin in tissue culture. They are classified as low-risk 
oncogenic viruses by the National Cancer Institute, and the viruses 
themselves must be handled under conditions equivalent to P2. Although 
there is no evidence that either virus causes tumors in man, SV40 
and related viruses do infect human beings and have been isolated in 
connection with several human disease states. Polyoma, however, 
does not infect man, and the Guidelines assume that polyoma inherently 
affords a higher level of biological containment. Therefore, more 
stringent physical containment is required for SV40 than for polyoma 
(Criteria 3, 5). 
The Guidelines require that any animal virus DNA used for recom- 
bination with a foreign DNA must itself be defective --that is, unable 
to propagate independently. 
