APPENDIX D— 17 
species that exchanges genes with E. coli, 
and under P2 + EKl conditions if not. 
(b) Purified cellular DNAs other than 
plasmids, bacteriophages, and other 
viruses. The formation of DNA recom- 
binants from cellular DNAs that have 
been enriched by physical and chemical 
techniques (i.e., not by cloning) and 
which are free of harmful genes can be 
carried out under, lower containment 
conditions than used for the correspond- 
ing shotgun experiment. In general, the 
containment can be decreased one step 
in physical containment (P4-»P3-^P2->- 
PI) while maintaining the biological con- 
tainment specified for the shotgun ex- 
periment, or one step in biological con- 
tainment (EK3->EK2-»EK1) while main- 
taining the specified physical contain- 
ment — provided that the new condition 
is not less than that specified above for 
characterized clones from shotgun ex- 
periments (Section (a) — iii) . 
(c) Plasmids, basteriophages, and 
other viruses. Recombinants formed be- 
tween EK-type vectors and other plasmid 
or virus DNAs have in common the 
potential for acting as double vectors 
because of the replication functions In 
these DNAs. The containment conditions 
given below apply only to propagation 
of the DNA recombinants in E. coli K-12 
hosts. They do not apply to other hosts 
where they may be able to replicate as 
a result of functions provided by the 
DNA inserted into the EK vectors. These 
are considered under other host-vector 
systems. 
(i) Animal viruses. P4+EK2 or P3EK3 
shall be used to isolate DNA recombin- 
ants that Include all or part of the 
genome of an animal virus. This recom- 
mendation applies not only to experi- 
ments of the “shotgun” type but also 
to those involving partially character- 
ized subgenomic segments of viral DNAs 
(for example, the genome of defective 
viruses, DNA fragments isolated after 
treatment of viral genomes with restric- 
tion enzymes, etc) . When cloned recom- 
binants have been shown by suitable bio- 
chemical and biological tests to be free of 
harmful regions, they can be handled in 
P3-I-EK2 conditions. In the case of DNA 
/Viruses, harmless regions include the late 
region of the genome; in the case of DNA 
copies of RNA viruses, they might In- 
clude the genes coding for capsid pro- 
teins or envelope proteins. 
(ii) Plant viruses. P3+EK1 or P2+EK2 
conditions shall be used to form DNA re- 
combinants that include all or part of 
the genome of a plant virus. 
(Itl) Eukaryotic organelle DNAs. The 
containment conditions given below ap- 
ply only when the organelle DNA has 
been purified' from isolated organelles. 
Mitochondrial DNA from primates: 
P3-f-EKl or P2-1-EK2. Mitochondrial or 
chlOTOplast DNA from other eukaryotes: 
P2+EK1. Otherwise, the conditions 
given under shotgun experiments apply. 
(iv) Prokaryotic plasmid and phage 
DNAs — Plasmids and phage from hosts 
that exchange genetic information ivith 
E. coli. Experiments with DNA recom- 
binants formed from plasmids or phage 
genomes that have not been character- 
See footnotes at end of article. 
ized with regard to presence of harm- 
ful genes or are known to contribute 
significantly to the pathogenicity of their 
normal hosts must use the containment 
conditions specified for shotgun experi- 
ments with DNAs from the respective 
host. If the DNA recombinants are 
formed from plasmids or phage that are 
known not to contain harmful genes, or 
from purified ' and characterized plasmid 
or phage DNA segments known not to 
contain harmful genes, the experiments 
can be performed with PI physical con- 
tainment + an EKl host-vector. 
Plasmids and phage from hosts that 
do not exchange genetic information with 
E. coli. The rules for shotgun experi- 
ments with DNA from the host apply to 
their plasmids or phages. The minimum 
containment conditions for this category 
(P2+EK2, or P3+EK1) can be used for 
plasmid and phage, or for purified ' and 
characterized segments of plasmid and 
phage DNAs, when the risk that the re- 
combinant DNAs will increase the patho- 
genicity or ecological potential of the 
host is judged to be minimal. 
Note.— Where applicable, cDNAs( i.e., 
complementary DNAs) synthesized in 
vitro from cellular or viral RNAs are in- 
cluded within each of the abovp classi- 
fications. For example, cDNAs formed 
from cellular RNAs that are not purified 
and characterized are included under (a) , 
shotgun experiments; cDNAs formed 
from purifi^ and characterized RNAs 
are included under (b) ; cDNAs formed 
from viral RNAs are included under (c) ; 
etc. 
3.' Experiments with other prokaryotic 
host-vectors. Other prokaryotic host- 
vector systems are at the speculative, 
planning, or developmental stage, and 
consequently do not warrant detailed 
treatment here at this time. However, the 
containment criteria for different types 
of DNA recombinants formed with E. coli 
K-12 host-vectors can. with the aid of 
some general principles given here, serve 
as a guide for containment conditions 
with other host-vectors when appropriate 
adjustment is made for their different 
habitats and characteristics. The newly 
developed host-vector systems should of- 
fer some distinct advantage 'over the 
E. coil K-12 host-vectors — for instance, 
thermophilic organisms or other host- 
vectors whose major habitats do not in- 
clude hiunans and/or economically im- 
portant animals and plants. In general, 
the strain of any prokaryotic species used 
as the host is to conform to the d^nition 
of Class 1 etiologic agents given in ref. 5 
(i.e., “Agents of no or minimal hazard. 
* * *.”) , and the plasmid or phage vec- 
tor should not make the host more haz- 
ardous. Appendix A gives a detailed dis- 
cussion of the B. subtilis system, the most 
promising alternative to date. 
At the Initial stage, the host-vector 
must exhibit at least a moderate level of 
biological containment comparable to 
EKl systems, and should be capable of 
modification to obtain high levels of con- 
tainment comparable to EK2 and EK3. 
The type of confirmation test(s) required 
to move a host-vector from an EK2-type 
classification to an EK3-type will clearly 
depend upon the preponderant habitat of 
the host-vector. For example, if the un- 
modified host-vector propagates mostly 
in, on, or around higher plants, but not 
appreciably in warm-blooded animals, 
modification should be designed to reduce 
the probability that the host-vector can 
escape to and propagate in, on, or around 
such plants, or transmit recombinant 
DNA to other bacterial hosts that are 
able to occupy these ecological niches, 
and it is these lower probabilities which 
must be confirmed. The following prin- 
ciples are to be followed in using the con- 
tainment criteria given for experiments 
with E. coli K-12 host- vectors as a guide 
for other prokaryotic systems. Experi- 
ments with DNA from prokaryotes (and 
their plasmids or viruses) are classified 
according to whether the prokaryote in 
question exchanges genetic information 
with the host-vector or not, and the con- 
tainment conditions given for these two 
classes with E. coli K-12 host- vectors ap- 
plied. Experiments with recombinants be- 
tween plasmid or phage vectors and DNA 
that extends the range of resistance of 
the recipient species to therapeutically 
useful drugs must use P3 physical con- 
tainment -|- a host-vector comparable to 
EKl or P2 physical containment + a 
host-vector comparable to EK2. Transfer 
of recombinant DNA to plant pathogens 
can be made safer by using nonreverting, 
doubly auxothrophic, non-pathogenic 
variants. Experiments using a plant 
pathogen that affects an element of the 
local flora will require more stringent 
containment than if carried out in areas 
where the host plant is not common. 
Experiments with DNAs from eukar- 
yotes (and their plasmids 6r viruses) can 
also follow the criteria for the corre- 
sponding experiments with E. coli K-12 
vectoi-s if the major habitats of the given 
host-vector overlap those of E. coli. If 
the host-vector has a major habitat that 
does not overlap those of E. coli (e.g., 
root nodules In plants) , then the contain- 
ment conditions for some eukaryotic re- 
combinant DNAs need to be increased 
(for instance, higher plants and their vir- 
uses in the preceding example) , while 
others can be reduced. 
4. Experiments with eukaryotic host- 
vectors — (a) Animal host-vector sys- 
tems. Because host cell lines generally 
have little if any capacity for propaga- 
tion outside the laboratory, the primary 
focus for containment is the vector, al- 
though cells should also be derived from 
cultures expected to be of minimal haz- 
ard. Given good microbiological prac- 
tices, the most likely mode escape of 
recombinant DNAs from a physically 
contained laboratory is carriage by hu- 
mans ; thus vectors should be chosen that 
have little or no ability to replicate in 
human cells. To be used as a vector in a 
eukaryotic host, a DNA molecule needs 
to display aU of the following proper- 
ties: 
(1) It shall not consist of the whole 
genome of any agent that is Infectious 
for humans or that replicates to a signif- 
icant extent in human cells in tissue 
culture. 
(2) Its functional anatomy should be 
known — that is, there should be a clear 
