NOTICES 
49603 
list of a defective genome or an appropriate 
conditional lethal mutant virus, making vec- 
tor and helper dependent upon each other 
for propagation However, this Is not a re- 
quirement 
In the case of very short Insertions. le„ 
of less than 50 base pairs. It Is not necessary 
that the recombinant be defective However, 
three requirements must be met. First, the 
Inserted sequence must be either (a) a prod- 
uct of In vitro synthesis: or (b) a sequence, 
or segment of a sequence, which has been 
purified * by chemical means or by cloning, 
and which does not code for a toxic product. 
Second. It must not be of viral origin. Third. 
It must be confirmed that the virus contain- 
ing the Inserted segment has not Increased 
in Its ability to replicate In human cells In 
tissue culture. 
Currently the only vertebrate viral DNAs 
that can be considered as meeting these re- 
quirements are the genomes of polyoma vi- 
rus. SV40. and Adenovirus types 2 and 5. 
(I) Polyoma tiruj. (a) Defective or Intact 
polyoma virus genomes, with appropriate 
helper. If necessary, can be used In P3 con- 
ditions to propagate DNA sequences from: 
Bacteria of Class 1 or Class 3 (See Appen- 
dix B). or their phages or plasmids, except 
for species of bacteria that produce potent 
polypeptide toxins * 
Eukaryotic organisms that do not produce 
potent polypeptide toxins • Whenever there 
Is a choice. It is urged that mouse cells, de- 
rived preferably from embroyos. be used as 
the source of eukaryotic DNA. Polyoma virus 
Is a mouse virus and recombinant DNA 
molecules containing both viral and cellular 
sequences are already known to be present 
In virus stocks grown at a high multiplicity. 
Thus, recombinants formed In vitro between 
polyoma virus DNA and mouse DNA are pre- 
sumably not novel from an evolutionary 
point of view 
(b) Polyoma genomes that are uncondi- 
tionally defective by virtue of a deletion In 
an essential gene region may be used, with 
appropriate helper. In P3 conditions to prop- 
agate. In mouse cell cultures, eubgenomlc 
segments of Class 1 or Class 3 viruses (See 
Appendix B) which do not replicate In 
mouse cells: provided. That It Is clear from 
the nature of the DNA segments to be com- 
bined that there Is no mechanism whereby 
a nondefective virus could be generated Viral 
DNA preparations used for ligation should 
contain lees than 1 % contamination with In- 
tact genomes 
It must be verified that the defective- 
virus helper-virus system has not been al- 
tered by the Inserted DNA so as to replicate 
In human tissue culture cells. 
(3) SV40 and adenovirus types 2 and 5. (a) 
Oenomes of 8V40 and Ad 3 and 5 which are 
unconditionally defective by virtue of a dele- 
tion In an essential gene region may be used, 
with helper, as vectors for DNA molecules 
of the types listed under Polyoma virus (See 
(1) above). Pt conditions are required. Viral 
DNA preparations used for ligation should 
contain less than 1% contamination with 
Intact genomes. 
(b) Such experiments may be carried out 
In P3 conditions If the Inserted DNA segment 
Is (1) a purified* segment of prokaryotic 
DNA lacking toxigenic genea or (11) a seg- 
ment of eukaryotic DNA whose gene product 
has been Identified, which does not code for 
a toxic product, and which has been previ- 
ously cloned In a prokaryotic host-vector sys- 
tem. or In P4 conditions as In (a), above. 
In the case of SV40. If helper Is used. It shall 
be confirmed that the defectlve-vlrus/helper- 
vlrus system does not reproduce significantly 
more viral DNA or progeny virus In human 
cells In tissue culture than does SV40, fol- 
lowing Infection at a multiplicity of Infec- 
tion of one or more helper SV40 viruses per 
cell; P3 conditions .may be used for this 
testing. 
(c) A viral DNA molecule which Is uncon- 
ditionally defective by virtue of a deleuon 
In the capsid genes and which Is capable of 
autonomous replication (analogous to a 
plasmid) can be used as a vector lor pro- 
pagating DNA from Claes 1 agents (See Ap- 
pendix B) in established cell lines P3 con- 
ditions can be ustd for shotgun type experi- 
ments: Provided. That the vector Is known 
to be free of the complete viral genome by 
virtue of having been propagated serially In 
Its absence. Otherwise. P4 containment con- 
ditions must be used 
(d) Recombinant DNA molecules consist- 
ing of SV40 or Ad. 3 or 5 DNA plus subgenom- 
Ic sequences from eukaryotic organisms or 
Class 1 or Class 2 agents (See Appendix B) 
may be used to transform nonpermlsslve 
cells under P3 conditions The recombinant 
molecule must be defective, and It must be 
demonstrated that no Infectious virus par- 
ticles are being produced: rescue of SV40 
from such transformed cells by co-cultiva- 
tion or transfection techniques must be car- 
ried out In P4 conditions. 
b. /nrevfebraf* Aosf-iecfor systems: Pesti- 
cide bacutoaruses Baculovlruses which have 
been registered by the Environmental Pro- 
tection Agency for commercial dissemination 
as biological pesticides have been suggested 
for use as cloning vectors. Two viruses are 
presently registered, the Heliothis sea bacu- 
lovtrua for control of the cotton boll worm 
and tobacco budworm. and the Orygia 
pseudotsugata baculovirus for control of the 
Douglas-fir tussock' moth’ These viruses 
have a host range which Is restricted to their 
natural hostis). which are serious Insect 
pests, they have been extensively tested for 
pathogenicity for many species of vertebrates 
and invertebrates, with completely negative 
results (28); they replicate best and cause 
natural disease only In the larval stage of the 
insect, thus simplifying containment; they 
have no taxonomic relatives among viruses 
of vertebrates, and tissue culture systems are 
available From available data, these proper- 
tles appear to offer great safety features. 
However, much still needs to be learned Par- 
ticularly needed Is Information on the na- 
ture of the host range specificity, and on the 
lnfectlvlty and persistence of the viral DNA 
In vertebrate and Invertebrate cell cultures. 
When such background Information Is 
available, and If It confirms the narrow host 
range specificity, a baculovirus vector may 
be used for cloning DNA segments derived 
from the host Insect, from another Environ- 
mental Protection Agency registered baculo- 
virus. or from an EKI bacterium using P2 
physical containment. During the explora- 
tory phases of this work It Is not envisioned 
that the cloning of other classes of DNA 
will need to be studied. 
c. Plant Aost-recfor systems. For cells In 
tissue cultures, seedlings, plant parts (e g., 
tubers, stems, fruits, and detached leaves) 
or whole-mature plants of small species (e g.. 
Arabidoprti) the descriptions of P1-P4 phys- 
ical containment conditions that have been 
previously specified are adequate. However, 
work with most whole plants pose additional 
problems The greenhouse facilities approxi- 
mating P2 laboratory physical containment 
conditions can be provided by: (1) Insect- 
proof greenhouses. (11) appropriate steriliza- 
tion of contaminated plants, pots. soil, and 
run-off water, and (111) adoption of the 
other standard practices for microbiological 
work. P3 physical containment can be suffi- 
ciently approximated by confining the oper- 
ations with whole plants to growth chambers 
like those used for work with radioactive Iso- 
topes. provided that (1) such chambers are 
modified to produce a negative pressure en- 
vironment with the exhaust air appropriately 
filtered. (U) other operations with Infectious 
materials are carried out under the specified 
P3 conditions, and (111) growth chambers 
are routinely fumigated and only used in In- 
sect-proof rooms In order to guard against 
Inadvertent Insect transmission of recom- 
binant DNA. The P3 and P3 conditions speci- 
fied earlier are therefore extended to Include 
these cases for work on higher plants. 
Whole plants or plant parts that cannot 
be adequately contained shall not be used as 
hosts for recombinant DNA experiments at 
this time. 
Organelle or plasmid DNAs or DNAs of vi- 
ruses of restricted ho6t range may be used 
as vectors. In general, criteria for selecting 
viruses similar to those given In the preced- 
ing section on animal systems are to apply 
to plant systems. 
DNA recom'-lnants formed between the 
Initial moderately contained vectors, and 
DNA from cells of species In which the vec- 
tor DNA can replicate, require P2 physical 
containment. However. If the source of the 
DNA Is Itself pathogenic or known to carry 
pathogenic agents, or to produce products 
dangerous to plants, or If the vector Is an 
unmodified virus, the experiments shall be 
carried out under P3 conditions. 
Experiments on recombinant DNAs formed 
between the above vectors and DNAs from 
other species may also be carried out under 
P2 containment If that DNA has been puri- 
fied • and determined not to contain harm- 
ful genes Otherwise, the experiments shall 
be carried out under P3 conditions If the 
source of the Inserted DNA Is not Itself a 
pathogen, or known to carry such patho- 
genic agents, or to produce harmful prod- 
ucts — and under P4 condltons If these con- 
ditions are not met. 
The development and use af host-vector 
systems that exhibit a high level of bio- 
logical containment, such as those using 
protoplasts or undifferentiated cells In cul- 
ture. permit a decrease of one step In 
the physical containment cpeclfled above 
( P4 -♦P3— * P2-* PI ) . 
d Fungal or similar lower eukaryotic host- 
vector systems The Containment criteria for 
experiments on recombinant DNAs using 
these host-vectors most closely resemble 
those for prokaryotes, rather than those for 
the preceding eukaryotes, since the host cells 
usually exhibit a capacity for dissemination 
outside the laboratory that Is similar to 
that for bacteria. Therefore, the contain- 
ment guidelines given for experiments with 
prokaryotic host-vectors (Sections III B-l 
and -2) provide adequate direction for ex- 
periments with these lower eukaryotic host- 
vectors. This Is particularly true at this 
time since the development of these host- 
vectors Is presently In the speculative stage. 
4 Complementary DNAs. Where applicable. 
cDNAs (l.e„ complementary DNAs) synthe- 
sized In vltso from cellular or viral RNAs 
are Included within each of the above clas- 
sifications. For example. cDNAs formed from 
cellular RNAs that are not purified and 
characterized are Included under Bl.a.. 
shotgun experiments; cDNAs formed from 
viral RNAs are Included under B l.b; cDNAs 
formed from purified and characterized 
RNAs are Included under B.l.c.; etc. Due 
to the possibility of nucleic add contamina- 
tion of enzyme preparations used In the 
preparation of cDNAs. the Investigator must 
employ purified enzyme .preparations that 
are free of viral nucleic acid. 
6. Handling recombinant DNA mole- 
cules. Preparations of recombinant DNA 
molecules which do not contain viable or- 
ganisms should be handled with good micro- 
biological techniques equivalent to those 
used In a PI laboratory. All recombinant 
DNAs should, however, be Inactivated prior 
to disposal. 
FEDERAL REGISTER, VOL 42, NO. 1 17 — TUESDAY, SEPTEMBER 27, 1977 
[ 171 ] 
