49602 
NOTICES 
specifications for Retroviruses In Section b. 
Similar considerations apply to attempts to 
clone viral genomes from exogenously In- 
fected cells; containment conditions are those 
for the viral genome In question, as defined 
In Section b. 
(2) Prokaryotic DNA recombinants. The 
minimum containment conditions consist of 
P2 physical containment-!- an EK1 host-vec- 
tor or P1 + EK2 and apply when the DNA Is 
from those bacteria that have been exten- 
sively characterized as to the physiological, 
biochemical, and/or genetic properties and 
when the risk that the recombinant DNAs 
will Increase the pathogenicity or ecological 
potential of the host Is Judged to be minimal. 
A description of the relevant properties of 
organisms which may meet these criteria 
should be submitted to the Office of Recom- 
binant DNA Activities, NIH, for approval of 
this minimal containment level by the Di- 
rector, NIH, on the recommendation of the 
Recombinant DNA Molecule Program Advis- 
ory Committee. 
Experiments with DNAs from bacteria that 
are not extensively characterized require P2 
physical containment-)- an EK2 host-vector or 
P3+EK1. Experiments with DNAs from path- 
ogenic species (Class 2 and plant pathogens. 
See Appendix B) must use P3 + EK2. 
b. Plasmids, bacteriophages, and other 
viruses. Recombinants formed between a 
vector and some other plasmid or virus DNA 
have In common the potential for acting 
as double vectors because of the replica- 
tion functions in these DNAs. The con- 
tainment conditions given below apply only 
to propagation of the DNA recombiants In 
E. coli K-12 hosts. They do not apply to 
other hosts in which the recombinants 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. 
(1) Viruses of eukaryotes* — (a) Viruses 
Of Warm-Blooded Vertebrates — 1. DNA Pi- 
ruses And DNA Transcripts Of Retrovirus 
Genomes. P4 physical containment-!- an EK1 
host- vector or P3 + EK3 shall be used for 
DNA recombinants produced with the whole 
genome or in shotgun type experiments with 
unpurlfled segments thereof. 
Containment can be reduced to P3 + EK2 
for clones shown to contain less than the 
complete potentially infectious viral genome, 
or for cloning purified 8 subgenomlc seg- 
ments.” However, In both cases, if the 
cloned segment contains any portion of a 
viral gene region that Is known or sus- 
pected to be responsible for neoplastic trans- 
formation, that portion must not be large 
enough to code for a functional polypep- 
tide. 
2. DNA Transcripts Of Other RNA Virus 
Genomes. In the case of RNA viruses with 
non-segmented genomes, containment con- 
ditions are as outlined In the preceding Sec- 
tion. 
In the case of RNA viruses with segmented 
genomes, the virtual Impossibility of syn- 
thesizing a single DNA molecule contain- 
ing the entire viral genetic Information from 
the extracted RNA permits a lower level 
of containment for shotgun type experi- 
ments. P3 physical containment + an EK2 
host-vector may be used. 
(b) Viruses Of Cold-Blooded Vertebrates . — 
1. DNA Viruses And DNA Transcripts Of Ret- 
rovirus Genomes. P4 physical containment 
+ an EK1 hostvector or P3 + EK2 shall be 
used for DNA recombinants produced with 
the whole genome or In shotgun type ex- 
periments with unpurlfled segments thereof. 
Containment can be reduced to P2 + EK2 
for clones shown to contain less than the 
complete, potentially Infectious viral genome 
or for cloning purified 8 subgenomlc seg- 
ments. However, In both cases, If the cloned 
segment contains, any portion of a viral 
gene region that Is known or suspected to 
be responsible for neoplastic transformation, 
that portion must not be large enough to 
code for a functional polypeptide. 
2. DNA transcripts of other RNA virus 
genomes. In the case of RNA viruses with 
non-segmented genomes, containment con- 
ditions are as outlined In the preceding 
Section. In the case of RNA viruses with seg- 
mented genomes, the virtual Impossibility of 
synthesizing a single DNA molecule contain- 
ing the entire viral genetic Information from 
the extracted RNA permits a lower level of 
containment for shotgun type experiments. 
P2 physical containment _)_ an EK2 host- 
vector may be used. 
(c) Viruses of invertebrates and proto- 
zoa. — 1. Baculoviruses which have been regis- 
tered by the Environmental Protection 
Agency as pesticides .’ For cloning all or part 
of the viral genome: PI physical contain- 
ment + an EK2 host-vector or P2 + EK1. 
2. Other viruses. Same containment levels 
as for viruses of cold-blooded vertebrates. 
(d) Viruses of plants. P3 physical con- 
tainment-!- an EK1 host-vector or P2-|_EK2 
conditions shall be used to form DNA re- 
combinants that Include all or part of the 
genome of a plant virus. 
(2) Eukaryotic organelle DNAs. The con- 
tainment conditions given below apply only 
when the organelle DNA has been purified 8 
from Isolated organelles. Mitochondrial DNA 
from primates: P3 physical containment-!- an 
EK1 host- vector or P2 + EK2. Mitochondrial 
or chloroplast DNA from other eukaryotes: 
P2 + EK1. Otherwise, the conditions given 
under shotgun experiments apply. 
(3) Prokaryotic plasmid and phage DNAs. 
The containment levels required for shotgun 
experiments with DNA from prokaryotes 
apply to their plasmids or phages. 
c. Lowering of containment levels for 
characterized or purified DNA preparations 
and clones. Many of the risks which might 
conceivably arise from some types of recom- 
binant DNA experiments, particularly shot- 
gun experiments, would result from the 
inadvertent cloning of a harmful sequence. 
Therefore, in cases where the risk of inad- 
vertently cloning the “wrong” DNA Is re- 
duced by prior enrichment for the desired 
piece, or in which a clone, made from a ran- 
dom assortment of DNAs, has been purified 
and the absence of harmful sequences estab- 
lished, the containment conditions for fur- 
ther work may be reduced. The following 
section outlines the mechanisms for such 
reductions. 
(1) Purified DNA other than plasmids, 
bacteriophages, and other viruses. The for- 
mation of DNA recombinants from cellular 
DNAs that have been purified 8 by physical 
and chemical techniques and which are free 
of harmful genes can be carried out under 
lower containment conditions than used for 
the corresponding shotgun experiment. 8 In 
general, the containment may be decreased 
one step In physical containment (P4-»P3_> 
P2-»P1) while maintaining the biological 
containment specified for the shotgun ex- 
periment. or one step In biological contain- 
ment (EK3-»EK2-»EK1) while maintaining 
the specified physical containment — pro- 
vided that the new containment condition is 
not less than that specified below for char- 
acterized clones from shotgun experiments. 
The institutional biohazards committee 
(IBC) may approve such a reduction. The 
IBC must notify the Office of Recombinant 
DNA Activities (ORDA) In writing of all such 
actions. 
(2) Characterized clones of DNA recombi- 
nants. When a cloned DNA recombinant has 
been rigorously characterized and there Is 
sufficient evidence that it is free of harmful 
genes, 3 experiments Involving this recombi- 
nant DNA may be carried out under lower 
containment conditions. 
(a) Institutional biohazards committees 
(IBCs) may give approval for a single step 
reduction In physical or biological contain- 
ment on receipt of evidence of characteriza- 
tion of the clone and Its probable freedom 
from harmful genes. The IBC must notify 
the Office of Recombinant DNA Activities 
(ORDA) In writing of all such actions. 
(b) Reduction of containment levels by 
more than one step will require prior ap- 
proval by ORDA. 
2. Experiments with other prokaryotic 
host-vectors. Host-vector systems which 
have been approved as HV1 systems may be 
used under P2 containment conditions for 
shotgun experiments with phages, plasmids, 
and prokaryotic DNA from non-pathogenlc 
prokaryotes which do not produce polypep- 
tide toxins* (i.e., organisms which can be 
cloned Into EK1 hosts under P2 conditions) . 
Other classes of recombinant DNA experi- 
ments with these HV1 systems will require 
prior approval and classification by NIH. 
Experiments with DNAs from eukaryotes 
(and their plasmids or viruses) will gener- 
ally follow the criteria for the correspond- 
ing experiments with E. coli K-12 host- 
vectors If the major habitats of the given 
host-vector overlap those of E. coli. The 
habitats of other host-vector systems should 
also be considered In relation to contain- 
ment. 
3. Experiments with eukaryotic host- 
vectors. — a. Vertebrate host-vector systems. 
Because this work will be done almost ex- 
clusively in tissue culture cells, which have 
no capacity for propagation outside the labo- 
ratory, the primary focus for containment 
Is the vector. Given good microbiological 
practices, the most likely mode of escape 
of recombinant DNAs from a physically con- 
tained laboratory is carriage by humans; 
thus vectors that have little or no ability 
to replicate in human cells should be chosen. 
Also, a recombinant virus should not lnad-. 
vertently pose a threat to any species. Prior 
to Its use as a vector In a vertebrate host 
cell system, a DNA molecule needs to display 
the following properties: 
(I) It shall not consist of the whole 
genome of any agent that Is Infectious for 
humans or that replicates to a significant 
extent In human cells In tissue culture. If 
the recombinant molecule Is used to trans- 
form nonpermissive cells (l.e., cells which 
do not produce Infectious virus particles), 
this Is not a requirement. 
(II) If the DNA Is of viral origin, the virus 
should not exhibit significant pathogenicity 
In Its natural biology, and the range of spe- 
cies In which the virus replicates efficiently 
should be narrow. 
(ill) Its functional anatomy shall be 
known — that is, there should be a clear idea 
of the location within the molecule of: 
(a) The sites at which DNA synthesis 
originates and terminates. 
(b) The sites that are cleaved by restric- 
tion endonucleases. 
(c) The template regions for the major 
gene products. 
(lv) It must be defective when carrying 
an inserted DNA segment; that Is, propaga- 
tion of the recombinant DNA as a virus must 
be dependent upon the presence of a com- 
plementing helper genome. Subject to the 
limitation In (1) above. It is not required 
In all cases that the recombinant be derived 
from a defective vector molecule; defective- 
ness resulting from Inactivation of an essen- 
tial gene by the inserted DNA sequence Is 
sufficient. If possible, the helper should 
either: (a) be integrated Into the genome 
of a stable line of host cells (a situation that 
would effectively limit the growth of the vec- 
tor to that particular cell line), or (b) con- 
FEDERAL REGISTER, VOL 42, NO. 187— TUESDAY, SEPTEMBER 27, 1977 
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