Gene, l(1977) 123-139 123
0 Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands

A SUMMARY OF THE NATIONAL INSTITUTES OF HEALTH (USA)
GUIDELINES FOR RECOMBINANT DNA RESEARCH

MAXINE F. SINGER
Laboratory of Biochemistry, National Cancer Institute. Bethesda. Maryland 2001 4 (U.S.A. )
(Received November lst, 1976)

(Accepted November 18th. 1976)

On June 23,1976, Donald S. Fredrickson, Director, National Institutes of
Health, announced publication of guidelines[ 11 designed to eliminate or mini-
mize any potentially hazardous consequences of what has been called recom-
binant DNA research. The guidelines were subsequently published in the
United States Government publication, the Federal Register (Part I1 for
7 July 1976).

The promulgated guidelines are the result of a year and a half of intensive
work by the NIH Recombinant DNA Molecule Program Advisory Committee*

*Recombinant DNA Molecule Program Advisory Committee (July 1,1976):
Stetten, De Witt, Jr. (Chairman), Deputy Director for Science, National Institutes of
Health, PW, Betheeda, MD 20014.
Adclberg, Edward A., -
New Haven, CT 06610.
Curtia, Roy, 111, -
Birmingham, AL 36294,
Darnell, James E. Jr., --
University, New York, NY 10021.
Day, Peter R., -
Station, New Haven CT 06604.
Helinski, Donald R., -
Sam Diego, La Jolla, CA 92037.
Hogness, David S., -
Stanford, CA 94306.
Kutter, Elizabeth, M.,-
College, Olympia, WA 98506.
Littlefield, John W., -
Baltimore, MD 21205.
Redford, Emmette S., -
Affairs, University of Texas, Austin, TX 78712.
Rowe, Wallace P., -
and Infeetiour Diseases, Bethesda, MD 20014.
Setlow, Jane K,-
Laboratory, Upton, Long Island, NY 11972.
Spizizen, John, -
Foundation, La Jolla, CA 92037.
Szybaleki, Waclaw, -
Wisconsin, Madison, WI 63706.
Gartland, William J. (Executive Secretary), National Institute of General Medical Sciences,
Bethesda, MD 20014.

Professor, Department of Human Genetics, Yale University,

Professor, Department of Microbiology, University of Alabama,

Professor, Department of Molecular Cell Biology, Rockefeller

Chief, Division of Genetics, Connecticut Agricultural Experiment

Professor, Department of Biology, University of California at

Professor, Department of Biochemistry, Stanford University,

Faculty Member, Department of Biophysics, Evergreen State

Chairman, Department of Pediatrics, Johns Hopkins University,

Ashbel Smith Professor, Lyndon B. Johnson School of Public

Chief, Laboratory of Viral Diseases, National Institute of Allergy

Senior Geneticist, Biology Department, Brookhaven National

Chairman, Department of Microbiology, Scripps Clinic and Research

Professor of Oycology, McArdle Laboratory, University of

124

(hereafter called the Advisory Committee) as well as consideration of a variety
of views expressed to the Director either in writing or at a public hearing in
February 1976. A summary of the history of the.development of the guidelines
as well as the various views expressed by many commentatore is given in the
Director's Decision Statement[ 11, which accompanied publication of the
guidelines.
For the purposes of the guidelines recombinant DNA experiments are de-
fined as those involving molecules that consist of different segments of DNA
which have been joined together in cell-free systems, 8126 which have the
capacity to infect and replicate in some host cell, either autonomoudy or as
an integrated part of the host's genome. Fig.1 depids a generalized mom-
birmt DNA experiment and definegcertain terms as they are used in the
guidelines.

1

.@=% 'Fomian' DNA

HOS~ Cell

Fig.1. A typical recombinant DNA experiment.

At the upper left is a cell containing chromosomal DNA and several small
independent genetic elements. These small independent DNA molecules are
isolated from the cell and serve as one portion of the recombined DNA, the
segment termed the vector. Such elements may be circula~ DNA molecules
such as plasmids, or viral DNAs, and they can be cleaved, as shown, by re-
striction endonuclease (or by other means) to yield linear duplex DNA
strands with either sticky ends, or ends that can be made sticky by appropn-
ate modification. In most experiments, the recombinant DNA will finally be
reinserted into cells of the same species &om which the vector was isolated.

125

It is the genetic information encoded in the DNA of the vector which dti-
mately will be responsible for the continued existence and replication of the
recombined DNA in the recipient cell. At the upper right of Fig.1 another
cell is shown as a rectangle. This cell will serve as the source of DNA to be
joined to the vector. This DNA is termed the foreign DNA, and the rectan-
gular cell can represent a cell from any living species. As shown here, the
foreign DNA might contain chromosomal DNA or independent DNA ele-
ments, or both. It too can be cleaved to yield fragments of various lengths
with sticky ends, or ends that can be made sticky. The foreign DNA frag-
ments are then joined to the vector DNA, usually by duplex formation at
the sticky ends, followed by closure of the internucleotide bonds with poly-
nucleotide ligase. The recombinant DNA is subsequently inserted into a
recipient cell, which is called the host. Again, the host cell will most likely
be of the same species as that used for the isolation of the vector. The cells
are then placed under conditions where either they or the recombined DNA
can replicate.

In the experiments discussed in the guidelines the host cells are generally
single living cells, either microorganisms such as bacteria, or animal or plant
cells grown as single cells in tissue culture.

General principles. The guidelines start with a statement of general princi-
ples and these are consistent with the general conclusions published in the
report of the International Conference on Recombinant DNA at Asilomar,
California, in February of 1976[2]. (I) The first principle is that there are
certain experiments which, in the light of currently available information,
may be judged to present potential hazards of so serious a nature, that they
should not be attempted at this time. (2) A large group of feasible experi-
ments appear to pose lesser or no potential hazard, and can therefore be per-
formed provided that the information to be obtained, or the practical bene-
fits anticipated, cannot be obtained by conventional methods, and provided
that appropriate safeguards for containment of potentially hazardous orga-
nisms are incorporated into the design and execution of the experiment.
(3) The more serious the nature of any possible hazardous event, the
more stringent should be the safeguards against escape of the potentially
hazardous agents. The safeguards should be at least as stringent as those
generally used to handle the most hazardous parent of the recombinant.
Since the potential hazards and their estimation are conjectural and specula-
tive, the levels of contahment required for potentially hazardous organisms
should be set high initially, and modified only when there is substantial rele-
vant information to advise such modifications. (4) The guidelines are to
be reviewed at least anndy in order to account for new information.
Containment methods. Three approaches to the problem of containing
potentially hazardous organisms form the basis of the safeguards recommended
by the guidelines. Each of the three may be viewed as setting up barriers to
the dissemination of potentially hazardous organisms from the laboratory
situation, and as setting up barriers between the laboratory worker and the

126

organisms. Two of these approaches involve the limitation of the actual physi-
cal escape of the organisms, and are referred to as physical containment. The
first such approach is the set of standard micro biological pmctices, that have
been developed over a period of many years, and are widely used for handling
pathogenic organisms both in research and clinical laboratories. In the hands
of well trained personnel, these procedures have proven to be effective in
safeguardhg both the worker and the environment from the spread of patho-
genic agents, The second approach to physical containment invol.ves the use
of special kinds of equipment and facilities (1) to limit spread of aerosols,
(2) for decontamination and containment of laboratory air and wastes, and
(3) limitation of access to laboratories. As with the standard microbiological
techniques the type of equipment and facilities are not new, but have been
developed and used previously for containment of known pathogenic orga-
nisms.
The guidelines go into some detail concerning the practices and facilities
required for physical containment: four levels of physical containment are
specified. They are termed P1, P2, P3 and P4 in the document, in the order
of increasing levels of containment. PI, the lowest level, consists of the use of
the standard microbiological practices mentioned before. The P2 and the next
higher level P3, each require special procedures and facilities (including verti-
cal laminar flow biological safety cabinets and laboratories maintained at
lower air pressure than the surrounding building) designed to limit to in-
creasing extents any possible accidental escape of potentially hazardous
organisms. Finally, P4, the maximum level of containment requires sophisti-
cated and isolated facilities designed for maximum containment. Each of the
levels, P2 through P4, assumes that the techniques demanded by P1, the
standard microbiological practices, will be followed. Furthermore, for each
level, relevant training of personnel is mandatory. The training is to include
the nature of the potential hazards, the technical manipulations, and instruc-
tion in the biology of the relevant organisms and systems. Specific emergency
plans, to be used in case of accident, are required and serological monitoring,
where appropriate, is to be provided.
The third approach to the problem of containing potentially hazardous
organisms within the laboratory is the use of biological barriers. Biological
containment is defined as the use of host cells and vectors with limited abili-
ty to survive outside of very special and fastidious laboratory conditions that
are unlikely to be encountered by escaped organisms in natural environments.
Biological containment is an integral part of the experimental design, since
the host and vector will need to be chosen, in any given experiment, with a
view both to the purpose of the experiment and to containment. The guide-
lines stress that physical and biologic containment procedures are comple-
mentary to one another each one serving to control any possible failure in
the other. The use of both in a given experiment affords much higher levels
of containment than either one alone. Therefore, the guidelines always re-
commend both tr particular level of physical containment, and a level of bio-

127

logical containment for any given experiment. The guidelines explicitly re-
cognize that novel techniques which enhance physical and biological contain-
ment capabilities are likely to be evolved as research proceeds and may reduce
the needs for the standard physical containment procedures. Such innovations
are to be considered as part of the on-going review of the guidelines for appro-
priate revision.

PU blication. The guidelines recommend that publications describing work
on recombinant DNA include a description of the containment procedures
used.

Experiments to be deferred. The first class of experiments described in the
guidelines are those which are not to be carried out at the present time. While
it may be argued that a combination of P4 physical containment and a high
level of biological containment could essentially contain these recombinants,
the magnitude of the possible dangers, were containment to fail, dictates that
these experiments be deferred. This class of experiments includes the follow-
ing.
(1) Any experiments in which a portion of the recombinant DNA derives
from pathogenic organisms listed under classes 3,4 and 5 of the document
entitled Classification of Etiologic Agents on the Basis of Hazard, as published
by the Center for Disease Control (CDC), United States Public Health
Service or from oncogenic viruses classified by the National Cancer Institute
as moderate risk. The CDC document categorizes naturally occurring orga-
nisms and viruses known to be pathogenic to man and agriculturally impor-
tant species on a scale of increasing hazard, going from 1 to 5. Class 5 agents
are excluded from the U.S. by law. Class 4 includes such agents as wild type
smallpox virus, wild type yellow fever virus, Herpesuirus simiue, and Lassa
virus. Class 3 includes Brucella, arboviruses and agents causing encephalitis,
psittacosis agents, Rickettsiae and vesicular stomatitis virus. Class 2 includes
agents which may produce diseases of varying degrees of severity if acciden-
tally inoculated into iaboratory workers, but which are considered normally
containable by standard laboratory practices. Examples of class 2 agents are
various species of Salmonella, agents causing amoebic dysentery, and mumps,
measles and rubella viruses. Class 2 agents may be used in recombinant DNA
experiments. The NCI classifies agents such as feline sarcoma and leukemia
viruses, and woolly monkey fibrosarcoma virus as moderate risk.

(2) Deliberate formation of recombinants containing the genes for toxins
of very high toxicity. Examples of this class are botulinus toxin or diphtheria
toxin, and venom from insects and snakes.
(3) Deliberate creation from plant pathogens of recombinant DNAs that
are likely to increase either the virulence of the pathogenic material or the
range of species susceptible to the disease.

(4) Certain of the possible beneficial applications of DNA recombinant
research involve the creation of organisms with the ability to carry out useful
environmental functions. Release of such organisms into the environment
may at some point be required to test their efficacy, and certainly to make

128

use of them. The guidelines state that deliberate release of any organisms
containing a recombinant DNA molecule is not to be undertaken at present.

to acquire it naturally if such acquisition could compromise the use of a drug
to control disease agents in human or veterinary medicine or agriculture.
(6) Experiments must be limited in scale to quantities of fluid less than

10 1 with recombinant DNAs known to make harmful products. The guide-
lines state that the Advisory Committee may make exceptions to this rule
for particular experiments deemed to be of direct societal benefit if appro-
priate equipment is used.

The use of bacterid hosts and uectora Recognizing the relation between
the host-vector system required by the experiment and the design of suitable
biological containment, experiments using the same hostcvector system are
grouped together. At present, the system of choice for many experiments is
the common laboratory bacterium, E. coli strain K12, and independent
genetic elements (plasmids and bacteriophage) known to reside or replicate
in this strain. There are several factors contributing to this conclusion.
Strain K12 has been studied extensively and can be readily manipulafed for
recombinant DNA experiments. This same extensive experience and ease of
manipulation permits modification of B. coli K12 and the vectors by classical
genetic techniques, for the purpose of establishing biological containment.
The guidelines also discuss arguments against the use of E. coli K12, in parti-
cular the intimate association of various other strains of E. coli with humans.
Far this reason the guidelines urge that efforts be made to develop alternate
bacterial host-vector systems. For this reason also, the guidelines recommend
the cautious use of E. coli K12 host-vector systems.
The nature and manner of achieving biological containment with this system
is described in the guidelines. E. coli K12 appears to be harmless itself, it does
not usually either establish itself in the normal bowel, or multiply significantly
in the alimentary tract. These facts suggest that accidental ingestion of a small
number of bacteria by a laboratory worker would not result in extensive
spread of the bacterium outside the laboratory. The normal situation may be
altered when people are either taking antibiotics, or have certain abnormal
digestive conditions and it is recommended that such individuals refrain from
work for the duration of the abnormal situation. However, while E. coli K12
does not establish itself as a growing strain in normal bowels, it does remain
alive during its passage through the tract. Therefore transfer of plasmid or
bacteriophage vectors containing foreign DNA from the original E. coli K12
host to bacteria resident in the ktestines or bacteria encountered after excre-
tion must be considered. The guidelines specify the use of nonconjugative
plasmids as vectors in recombinant research, because they cannot promote
their own transfer. However transfer of a resident nonconjugative plasmid is
possible in nature if the recombinant-containing host acquires a conjugative
plasmid that is derepressed for transfer. For any given host-plasmid combina-
tion used in a recombinant DNA experiment it will be necessary to assess the

(6) Transfer of a drug resistance trait to microorganisms that are not known

129

possibilities for transfer of the recombinant DNA in order to evaluate the
degree of biological containment. While we are missing some relevant infor-
mation, the available data suggest that the probability of transfer can be quite
low, depending on the particular nonconjugative plasmid used, on whether
or not the conjugative plasmid is repressed with respect to expression of
donor fertility, and on the viability of the host cell in natural environments.
With certain known and useful plasmids, triparental matings involving first
the acquisition of a conjugative plasmid and second, transfer of the noncon-
jugative plasmid to a third cell occur at frequencies that are less than one in
lo9 and in fact are usually undetectable under laboratory conditions designed
to resemble natural conditions. Hostcvector systems made up of E. coli K12
and such plasmids thereiore appear to have only very limited ability to spread
recombinant DNA molecules.
Analogous considerations apply when bacteriophage are to be used as vec-
tors for foreign DNA. Bacteriophage vectors could be spread either as mature
phage, or in cells either lysogenic for the phage or carrying the phage as a
plasmid. The bacteriophage lambda can be used to illustrate relevant considera.
tions since this widely studied bacteriophage is most likely to be used for re-
combinant experiments at the present time.
Considering first escape as a phage particle, lambda is sensitive to the
acidity of the stomach and is likely to be destroyed there. Normal intestinal
strains of E. coli are usually not susceptible to infection by lambda and in
fact, susceptible strains are rare in nature. Further, in at least one case, inges-
tion of 10" lambda particles yielded no detectable lambda in resulting feces.
Lambda is also readily destroyed by drying in air, DisseminaVan of lambda
recombinants through lysogen formation, a frequent event with susceptible
E. coli strains, can be minimized by use of mutant varieties of lamba which
lack genes necessary for lysogen formation: with such phage the frequency
of integration into the host chromosome is reduced to       Finally,
conversion of lambda DNA to a stable plasmid is also a relatively unlikely
event, occurring at a frequency of about

Considering then the properties of E. coli K12, as well as those of the
existing plasmid and bacteriophage vectors, the proposed guidelines conclude
that, using such hosbvector systems, recombinant DNAs are unlikely to be
spread by the ingestion or dissemination of the few hundred or thousand
bacteria, such as might be involved in laboratory accidents, given standard
microbiological practice. Therefore, these existing systems, and analogous
combinations of E. coli K12 with other vectors and bacteriophages are judged
to offer a moderate level of biological containment and are defined as EK1,
the lowest level of biological containment for experiments with E. coli
systems. Other prokaryote host-vector systems need to be evaluated using
the same general principles as those applied to the E. coli K12 situation.
As with physical containment levels, increasing numbers specify increasing
levels of biological containment for E. coli systems. The next level is called
Em. EM host-vector combinations must be demonstrated to provide a high

or

180

level of biological containment by suitable laboratory tests. They are ob-
tained by genetic modification of either E. coli K12 host cells or the relevant
plasmids and bacteriophage or both. More specifically, the guidelines state
that in order to qualify as EK2 the modified system composed of derivatives
of E. coli K12 combined with a particular vector should not permit Survival
of a genetic marker carried on the vector in other than specially designed
laboratory environments at a frequency greater than   Various examples
of the types of necessary modifications are suggested in the guidelines. For
example, modifiitions of the host might be mutations which result in
special nutritional requirements for growth or sensitivity to naturally oc-
curring materials such as bile salts, or elimination of nost-controlled restric-
tion and modification. Suggested modifications of plasmid vectors include
mutations making essential plasmid functions sensitive to normal body tem-
peratures or dependent on a specific host. Mutations which make native
phage particles containing a recombinant DNA unstable in natural environ-
ments and therefore unlikely to infect new E. coli cells should they escape
can be considered.

One additional level of contained E. coli hosbvector systems is defined in
the guidelines and is called EK3. EK3 systems are EK2 systems for which the
specified containment properties have been demonstrated not only by micro-
biological and genetic analysis but by appropriate tests in animals including
humans or primates and other relevant environments.

NIH, after evaluation and recommendation by the Advisory Committee.
Detailed data on the relevant properties of the system must be submitted for
consideration by the Committee. Thus far (January, 1977) the following
EK2 systems have been certified: E. coli strain ~1776 with either plasmid
pSClOl or plasmid pCR1, a derivative of colEl, and one lambda phage derivative
[ 31. Several other phage lambda derivatives, to be used in conjunction dth
specified partially disarmed E. coli K12 host cells, are under consideration by
the Committee. No EK3 systems have been submitted fDr certification as yet.
Information concerning certified systems and their availability can be obtained
from the office mentioned in ref.1 [4].

EK2 and EK3 host vector systems must be certified as such by the Director of

Classification of experiments currently pennissible with E. coli K12 host-
vector systems

Having defined the several levels of physical containment and biological
containment the specific recommendations for experiments using the E. coli
K12 host-vectorsystems can be described. Each type of experiment is assigned
both a physical containment level, that is a P level, and a biological contain-
ment level, that is an EK level and the particular combination of the two
reflects the severity of the estimated potential hazard. The Guidelines are
organized, for the E. coli systems, according to the source and nature of the
foreign DNA, as outlined in Fig.2. A sample of DNA containing essentially

131

Experiments with E. coli` "Host-Vector" Systems
Nature of the `Foreign' DNA
       I Total DNA of an organism I

Mixture of
all fragments
"shotgun"

fragment (99%)
Frez of harmful

Extrachromosomal DNA
   (purified)

1--.1- ---I- - 7-

bacteriophage

Organelle

- --

I--- -

IEllrhangerl Exchanoer

I I I I

Fig.2. Source and nature of the foreign DNA.

all the genetic information of an organism can be isolated and fragmented.
If the experiment involves such a mixture of DNA fragments, it is referred
to as a "shotgun" and will call for a certain level of containment. Experiments
involving such mixtures of DNA fragments are assumed to be of relatively
high potential hazard because of the greater likelihood of unknown and therefore
maybe hazardous genes being introduced into a recipient cell compared to experi-
ments with a single, highly purified fragment. Purified fragments containing
mainly genes whose properties are known and are not harmful, offer less
potential hazard than a shotgun experiment. In some instances the foreign
DNA will itself be derived from extrachromosomal genetic elements. Such
extrachromosomal elements include the DNA of animal viruses, plant viruses,
other eukaryote organelles such as mitochondria and chloroplasts, as well as
prokaryote plasmids or bacteriophages, of the same type used as vectors.
Each of these cases is treated separately in the guidelines. The prokaryote
sources are treated differently, depending on whether the source of the
"foreign" DNA is an organism that does or does not exchange genetic infor-
mation with E. coli in nature.

Guidelines for experiments with E. coli host-vector systems. Table I shows
the containment required for shotgun experiments when the foreign DNA is
a mixture of fragments derived from eukaryotes. The physical and biological
containment is listed for various possible DNA sources: both must be used as
they complement each other. For example, DNA from primates requires the
most stringent containment, since the estimated potential hazard either from

132

TABLE I

Guidelines for experiments with E. coli hort-vector systems using foreign DNA derived
from eukaryotes. "Shotgun" experiments

Foreign DNA source

~~~

Physical Biological

Primates

Embryonic
Other mammals
Birds
Cold-blooded vatebrates

Embryonic
Toxin
Lower eukaryotes
If pathogenic or toxigenic
Manta
If pathogenic or toxigenic

P3
P4
P3
P3
P8
p2
P2
'P3
P2
P3
P2
P3

EK3
EK2

or

EK2
EK2
EK2
EK2
EK1
EK2
EK1
EK2
EKl
EK2

genes that might function in humans with untoward effects, or from pathoge-
nic viral DNAs residing in primate tissue is judged to be most serious. The ex-
periments require either P3 and EK3, or P4 and EK2, and it should be re-
called that only the latter combination, P4 and EK2, is feasible at present
and even then only at the limited number of P4 facilities. Another point of
interest is that in two instances, primates and cold blooded vertebrates, con-
tainment requirements are lower if the DNA is ieolated from embryonic tie-
sue, or germ line material, since such material is less likely to be contaminated
by pathogenic viruses or other adventitious agents than is adult tissue. Thus,
if the foreign DNA is tom cold-blooded vertebrates, P2 and EK2 are required
but P2 and EKl can be used if the DNA is from embryonic or germ line tis-
sues. If the cold blooded vertebrate is known to produce a potent toxin, P3
and EK2 must be used. In some instances, lower eukaryotes for example, the
guidelines require more or less stri   conditions depending on whether or
not the source of foreign DNA is known
might be infected with a pathogen, or is

Table I1 summarizes the guidelines for shotgun experiments when the
source of the DNA is a prokaryotic organism. First, those prokaryotes which
are known to exchange genetic information with E. coli in nature are con-
sidered. The containment requirements are low for this group and vary with

athogenic or toxigenic, or
to make a harmful product.

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TABLE 11

Guidelines for experiments with E. coli host-vector systems using DNA derived from
prokaryotes. "Shotgun" experiments

Foreign DNA lource Physical Biological

-~~~

~

Prokaryota.

"Exchangers"

class 1 (CDC)'
ClaS6 2 (CDC)
Plant pathogens

"Non-exchanged'
Clam 1 (CDC)

Clam 2 (CDC)
Plant pathogen

P1
P2
P2

EK1

EK 1 -EK2b

EKI-EK~~

EK2
EK1

P2
P3
P3 EK2
P3 EK2

or

' CDC refers to the claseification of Etiologic Agents on the Basis of Hazard, as Published

by the Center for Disease Control (CDC), United States Public Health Service.
EK1 or EK2, depending on whether the species is of low or moderate pathogenicity.

that recombination experiments will create new genetic corn binations, never
tested by nature. When prokaryote donors not known to exchangs DNA
with E. coli in nature are used, however, there is a greater potential for new
genetic combinations to be formed and expressed.
Characterized clones obtained from shotgun experiments may not be as
potentially hazardous as the original mixture of cells. Cloning of the reci-
pient host cell containing the DNA fragment of interest will be one of the
normal aims of any recombinant experiment, The guidelines state that when
a clone has been obtained from a shotgun experiment, and has been rigorous-
ly characterized, and when there is sufficient evidence that it is free of harm-
ful genes, then experiments involving the clone can be carried out under P1
and EKl conditions if the foreign DNA was from a species that exchanges
genes with E. coli in natme, and under P2 and EX1 conditions if it does not.
Similarly, when the initial recombination involves a purified segment of
the foreign chromosomal DNA, rather than a mixture, the potential for
growth of a hazardous organism will be le=, since 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% of the total DNA in the preparation, by weight, and
further, they require-evidence that no harmful genes are present. Under such
circumstances {he 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 one step in biological contain-

134

ment. Thus, for example, shotgun experiments with DNA from birds require
P3 and EKZ. A DNA fragment from birds that is ftee from harmful genes,
and purified to 99% purity prior to joining to a vector, would require either
P2 and EK2 or P3 and EKl.
The final group of experiments utilizing E. coli host-vector systems that
are considered are those in which the foreign DNA is itself from an extra-
chromosomal element. As indicated in Fig.2 it is assumed that such DNA is
purified away from chromosomal DNA prior to recombination. Various pot+
sible sources of extrachromosomal DNA are listed in Table III and the recom

TABLE III

Guidelines for experiments with E. coli host-vector systems in which the foreign DNA
is from an extrachromosomal element

Containment

"Foreign" DNA source

~~~

Physical

Biological

Animal viruses

If cloned, free of
harmful genes

Plant viruses

Eukaryote organellesa

Primates
Other

Prokaryote (plasmids and phage)

"Exchangers"
Non-pathogene
Pathogen
" Non-exchangers"

or

P4
P3

P3

P3
P2

or

or

P8
P2
P2

P1
As for shotgun
As for shotgun

EK2
EK3

EK2

EK1
EK2

EK1
EK2
EKl

EK1

a The organelle DNA must be purified from isolated organelles. Otherwise conditions
indicated for shotgun experiments apply.

mended combined containment given. For example, DNA from all or part
of the genome of an animal virus requires P4 physical containment and an
EK2 host vector system, or, alternatively, P3 and EK3. When the recombi-
nants have been purified by clonirig, and shown to be free of harmful regions
of the viral genome, then experiments can be moved to P3 and EK2,
When complementary DNAs (cDNA), synthesized in vitro from RNA pre-
parations, are used in recombination experiments, the c
ments are as described for isolated DNA preparations.
the cDNA is less than 99 per cent pure, shotgun conditions are required.

135

Guidelines for experiments with animal host-vector systems. Many recom-
binant DNA experiments will involve the use of systems in which the host
cells are eukaryote cells grown as single cells in tissue culture: useful vectors
may include extrachromosomal DNA elements such as orgmelle 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. The cells themselves 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 considerations focus on the
viruses. Animal viruses can escape a laboratory in a viable form, especially
if laboratory workers become infected. There are two animal viruses whose
DNAs are, now, technically useful as vectors; polyoma and simian virus 40
(SV40). The cleavage of these molecules with restriction endonucleases has
been studied entensively. In their respective normal hosts, mouse for polyoma,
rhesus monkeys for SV40, neither virus causes a known disease. Polyoma does
not infect human cells grown as single cells in the lab and also does not appear
to infect humans, since humans exposed to polyoma do not produce anti-
bodies. SV40 does infect both human cells grown as single cells in the labora-
tory, and whole human beings, as evidenced by the active production of anti-
bodies and the reports of isolation of SV40 from humans. "his virus conta-
minated the early Salk polio vaccines and millions of people were inadvertent-
ly inoculated with it in the middle 1950s. To date, there is no indication that
the recipients of the vaccine suffered any related difficulty. Both polyoma and
SV40 are oncogenic, that is they cause tumor formation in newborn small
laboratory mammals, and both can transform a variety of cells of mammalian
origin. They are classified as low risk oncogenic viruses by the National Cancer
Institute, and the viruses themselves must be handled under conditions equi-
valent to P2. Because SV40 infects human beings, and also because SV40
and related viruses have been isolated in connection with several human
disease states, the proposed guidelines assume that polyoma inherently af-
fords a higher level of biological containment: therefore more stringent phy-
sical containment is required for SV40 than for polyoma.
The guidelines require that the viral DNA used for recombination with a
foreign DNA must itself be defective, that is, its propagation as a virus must
be dependent on the presence of helper virus which supplies the genes for the
missing unctions. This helper can be another defective or an appropriate con-
ditional lethal mutant virus. Mternatively the helper might be previously
integrated into the genome of a stable line of host cells. The use of a non-
defective genome as a helper is permissible if the alternatives are unavailable.
In certain kinds of experiments, no production of viral particles is required,
and no helper may be needed: biological containment is inherently greater in
the absence of virus particles since, as pointed out before, cells themselves
are relatively easy to contain.

required physical containment for these experiments, as summarized in

With these spec& of biological containment in mind, the guidelines specify

186

Table IV. The particular levels of physical containment depend on the source
of the foreign DNA, whether defective polyoma or defective SV40 is the
chosen vector, and finally on whether or not virus particles are produced.

Considering first experiments with defective polyoma vectors, under con-

TABLE IV

Guidelines for use of polyoma and SV40 as vectors

Physical
containment

"Foreign" DNA

Polyoma SV40

With virion production

Non-pathogen

Low pathogenicitv

If purified, cloned, harmless

P3
P3
P4

P4
P3

-

No virion production

Non-pathogen                    P3 P3
Low pathogenicity                 P3

-

ditions where viral particles are produced ... if the foreign DNA is from a
nonpathogenic agent, P3 conditions are required, even if the DNA fragment
was purified first and does not contain harmful genes. If the foreign DNA is
from an organism with low pathogenicity, P4 must be used -until such time as
suitable tests indicate that only harmless genes are present and then experi-
ments can be continued at the P3 level. Still considering polyoma vectors, P3
conditions are required for experiments in which no Virus particles are pro-
duced. When defective SV40 is the vector, and virus particles are produced,
P4 conditions must be used and the foreign DNA must be from a nonpatho-
genic organism. Experiments can be done in P3 only after extensive and specified
kinds of purification of the DNA and demonstration that no genes for toxic
products are present. SV40 can not be used at all for experiments with DNA
from pathogenic organisms. When no SV40 virus particles are produced, ex-
periments with recombinants derived from nonpathogenic agents can be
carried out in P3 conditions.
Guidelines for experiments with plant host vector systems. The Guidelines
also contain recommendations for experiments in which plant cells will sefve
as hosts for recombinant DNA. The cells might be single plant cells grown
under laboratory conditions, or seedlings, plant parts, or small whole plants.
This is in fact the only instance where the guidelines address the question of
recombinant DNA experiments with whole organisms. Directions are given
for modification of the specifications for P1, P2, and P3 physical contain-
ment in order to provide conditions appropriate for work with plants.

Vectors for use in experiments with plants include plant organelle ENA

137

such as the DNA of chloroplasts, and DNA of viruses of low pathogenicity
and restricted host range. These vectors offer moderate levels of biological
containment, and the guidelines specify the physical containment levels out-
%ked in Table V. As before, the requirements are organized according to the

TABLJ3 V

Experiments with plant host-vector systems

-

Physical

Source of "foreign" DNA Containment

Species in which vector can replicate

If harmful products possible

P2
P3

Other species

Foreign DNA purified (99%), and

Foreign DNA not purified, and no

no harmful genes P2

harmful genes P3

Foreign DNA contains harmful genes

P4

source of the foreign DNA. If the foreign DNA is derived from a species in
which the vector DNA is known to be able to replicate, P2 conditions are re-
quired, unless the source of foreign DNA is pathogenic or produces products
dangerous to plants ... then P3 is required. If the foreign DNA is derived from
a species in which the vector is not known to replicate then more stringent
requirements govern and vary from P2 to P4 depending on whether the DNA
is purified, and whether it contains harmful genes.
Other host-vector eystems. Theoretically, there are a variety of organisms,
both prokaryotes and lower eukaryotes such as fungi and yeast which will be
interesting and useful hosts for experiments with recombinant DNAs. Some
may offer the special advantage of not infecting humans, animals or important
ecological niches. However, a variety of technical developments are needed
before useful vectors are available for these systems. The growth characteris-
tics of such hosts indicate that containment problems will be like those for
E. coli K12 hosts. The guidelines urge development of these systems and
point out that the detailed recommendations made for E. coli K12 systems
can be used as a guide in determining biological and physical containment
requirements for these syste,as when that is required.

defining the roles and responsibilities of individuals and institutions in
assuring compliance with required containment levels. The procedures, as
described, are primarily directed at grantees of the National Institutes of
Health. Similar procedures are in force for work carried out within the NIH
laboratories themselves, and for work carried out under contract arrange-
ments with the NIH.

Implementation of the Guidelines. The guidelines contain a large section

iaa

The principal investigator is required to assess any potential biohazards, to
institute appropriate safeguards and procedures, to minimize effects of possi-
ble accidents by planning, to train and inform all personnel, to report any
serious or extended illness of a worker or any accidents, and all of these must
be carried out on a continuing basis. Thus, the primary responsibility for con-
ducting experiments according to the guidelines is in his hands. Further, in
applying for grants to carry out experiments with recombinant DNA, the
investigator must include an estimate of the potential biohazards as well as
a statement as to the containment procedures that will be used. The applica-
tion must include certification as to the existence and availability of appro-
priate facilities, procedures, and training. The guidelines indicate that institu-
tions in which recombinant DNA experiments are carried out must establish
biohazard committees which can serve to examine equipment and facilities
and certify their compliance with the requirements. Such committees will
also serve as a source of advice and reference on physical containment facili-
ties, on properties of biological containment, and on training of personnel.
According to the proposed guidelines review of the certification and of
the investigator's judgment concerning the extent of potential hazard and
the required containment would be by NIH study sections, during the normal
scientific review of the application. The guidelines leave flexible the question
of resolving any differences between the evaluation of the investigator and
that of the study section. The guidelines do state, however, that in instances
where resolution of differences cannot be made, the matter should be re-
ferred to the Advisory Committee or the NIH Office of Recombinant DNA
Activities.
Application of the guidelines to work not supported by the National
Institutes of Health. Several agencies of the U.S. government other than the
National Institutes oi' Health provide support for biological and medical
research. Some of these agencies are cunently, or may in the future, sponsor
recombinant DNA experiments. Adoption of the NIH guidelines is being con-
sidered by these agencies. At this writing (January, 1977) the following have
stated that the Guidelines will be applicable to their grantees: the National
Science Foundation, the Energy Research and Development Administration,
the National Aeronautics and Space Administration, and the Department of
Defense.

Efforts are also underway to develop appropriate and effective ways to
extend the requirements in the guidelines to research supported by private
funds. In the meanwhile it is anticipated that voluntary compliance by the
private sector will be extensive.
Guidelines in other countries. The United Kingdom [ 61 has independently
developed guidelines for research with recombinant DNA. Comparison of the

tainment categories

what more stringent definitions than the NIH guidelines for the lower levels

139

of physical containment (P1 through P3): the NIH P4 specifications appear
to be more stringent than the comparable level in the British document. No
experiments are specifically prohibited by the British Report.
More precise comparison will be possible as specific experiences accumu-
late[S 1.

1

Copies of this document may be obtained from the Executive Secretary, Office of

Recombinant DNA Activities, Building 31, National Institutes of Health, Bethesda,
Maryland, 20014, (U.S.A.).
Berg, P., Baltimore, D., Brenner, 8. and Singer, M.F., Science 188 (1975) 991; Nature,
225 (1975) 442; Roc. Natl. Acad. Sci. USA, 72 (1975) 1981.
Enquist, L., Tiemeier, D., Leder, P., Weisberg, R., Sternberg, N., Nature, 259 (1976)
596-598.

Scientists involved in recombinant DNA research may keep informed of further develop-
ments concerning host-vector systems as well as other aspects of the field by subscribing
to the "Nucleic Acid Recombinant Scientific Memoranda". Inquiries should be addressed
to: Dr. E.C. Chamberlayne, Project Officer, FIC. Building 31, Room 2C15, National
Institutes of Health, Bethesda, Maryland, 20014 (U.S.A.).

Report of the Working Party on the Practice of Genetic Manipulation, June, 1976 London,
Her Majesty's Stationery Office.
A detailed review of activities in many countries has been prepared by John Tooze,
European Molecular Biology Organization, 69, Heidelberg, Germany.

2

3

4

6

6

Communicated by W. Szybalski.