IU-A-:-^aMeM2Ma). PI physical 
containment + an EK1 host- vector 
shall be used tar DMA recombinants 
produced with purified subgenomic 
^ ICfflPiOU.( Ji ) 
pa physical 
contain meat + mi EK1 host and a 
vector certified tor use In an EK2 
system of P3 + EK1 shall be used for 
DNA recombinants produced with tti 
cDHA copies of the whole genome. or 
(11) purified cDBA copies of viral 
mRNA-(JT) 
III-A-2-a-dMdl. Dou ble-Stra nded 
Segmented RNA Virtue*. Pt physical 
Tilnsp lit + an EK1 host- vector 
shall be used for DNA recombinants 
produced with (1) mixtures ctf subgewo- 
mlc cDNA segments. (11) a specific sub- 
g monte cDNA segment, or (111) puri- 
fied cDNA cup tos of viral mRNA. (17) 
m-ArJ+UMd. RNA Plant Viruses 
and Plant VI rolls. Pi physical con- 
tainment + an SKI host- vector shall 
be used for DNA recombinants pro- 
duced with (I) eDNA copies of the 
admit Nrxi Rename. (11) aubgenoode 
cDNA segments, or (111) purified cDIVA 
copies of viral mRNA.( J7) 
ITT-A-J-a-CX). Intracellular Vital 
DNA. Physical and biological contain- 
ment specified tor shotgun experi- 
ments with eukaryotic cellular DNA 
[see section IIJ-A-UMa)! shall be 
used tor DNA recombinants produced 
with Integrated Aral DNA at viral gen- 
omes present In infected cells. 
LU — A-2-b. fXJcaryottc Organelle 
DNA'*. pa phyMssl containment + an 
EK1 host-vector, or PI + EK2. for mi- 
tochondrial or chloroplast DNA from 
eukaryotes when the organelle DNA 
has been obtained from Isolated organ- 
elles. Otherwise, the conditions given 
for shotgun expertmeufts apply. 
m-A-2-c. Prokaryotic Plarmld and 
Phage DNA 'a The containment l evels 
required for shotgun e xp e rim e nts with 
DNA from prokaryotes apply to their 
plasmids or phagea. 
III-A-3 Lowering d Comlainmewt 
Level * for Characterized or Purified 
DNA Preparation* and Clone*. Many 
of the risks which might conceivably 
arise from some types of recombinant 
DNA experiments, particularly shot- 
gun experiments, would result from 
the lnadvertewt cloning of a harmful 
sequence. Therefore. In cases where 
the risk of Inadvertently cloning the 
"wrong" DNA is reduced by prior en- 
richment for the desired piece, or In 
which a done, made from a random 
assortment of DNA's, has been puri- 
fied and the absence of harmful se- 
quences established, the containment 
conditions for further work may be re- 
duced. The following section oatlines 
the mechanisms for such reducttons. 
III-A-3-a. Purified DNA Other than 
Plasmid*. Bacteriophage*, and Other 
Viruses The formation of DNA recom- 
binants from cellular DNA's that have 
been purified (41) and which are free 
of harmful genes(J) can be carried out 
under tower containment conditions 
than used for the corresponding shot- 
gun experiment. (42) The containment 
may be decreased one step In physical 
containment ( P4 P3 -* P2 -» PI) 
while maintaining the biological con- 
tainment specified for die shotgun ex- 
periment or woe step In biological con- 
tainment EK3 — EK2 — EK1) while 
maintaining the specified physical 
containment. The Instftutlonal biosa- 
fety committee (IBC) must review and 
may approve such a reduction. The 
IBC must notify the NIH Office of Re- 
combinant DNA Activities (ORDA) In 
writing of all such actions. IBC ap- 
proval is sufficient for such a reduc- 
tion except for (1) primate DNA which 
also requires prior NIH approval [see 
section IIl-A-l-a-<l)] or (11) any lower- 
ing at containment under section III- 
A-3-a to levels below P1 + EK1. which 
also requires prior NIH approval. 
IITA-3-b Characterized Clone* of 
DNA RecauntinunU. When a cloned 
DNA recombinant has been rigorously 
characterized and there is sufficient 
evidence that R is free of harmful 
genes. (J) experiments Involving this 
recombinant DNA may be carried out 
under lower containment conditions, 
as described below. 
III-A-i-b-( 1 ). Institutional biosafety 
committees (IBCi) map give approval 
for a single-step reduction In physical 
or biological containment on receipt of 
evidence of characterization of a clone 
derived from a shotgun experiment 
and Its probable freedom from harm- 
ful genes. The IBC must notify ORDA 
In writing of all such actions. IBC ap- 
proval is sufficient for such a reduc- 
tion excer* lor (T) primate DNA. which 
requires prior NTH approval tsee sec- 
tion LIl A-l-v-( 1H. or (tl) any lowering 
of containment under section TTT-A-3- 
b to levels below P1 + E3C1. which also 
requires prior NIH approval. 
in-A » b-(lt Reduction of contain- 
ment levels by more than one step or 
cases Involving primate DNA. or cases 
involving lowering of containment 
under section III-A-3-b to levels below 
Pl + EKl. will require prior approval 
by NIH. 
IH B Experiments with Other Pro - 
kan/otic Hoit-Vector*. 
m-B-1. HVl system*. Host-vector 
systems which have been approved as 
HVl systems may be used under P2 
containment conditions for shotgun 
experiments with phages, plasmids, 
and DNA from nonpathogenic prokar- 
yotes which do not produce polypep- 
tide toxlns.(Ji) 
Other classes of recombinant DNA 
experiments with these HVl systems 
will require prior approval and classifi- 
cation by NIH. Experiments with 
DNA's from eukaryotes (and their 
plasmids or viruses) will generally 
follow the criteria for the correspond- 
ing experiments with E. coli K-12 
host vectors tf the major habitats of 
the given host-vector overlap those of 
£1 calx. The habitats of other host- 
vector systems should also be consid- 
ered In relation to containment. 
III-B-2. Return of DNA Segment* to 
Non-HVl Host of Origin. Many of the 
prokaryotes that exchange genetic In- 
formation with E. coli by known phys- 
iological processes are expected to be 
exempt from these Ouidelines by ap- 
pearing on the "list of exchangers" 
(see Section I-E-4). For a prokaryote 
which can exchange genetic Informa- 
tion (J5) with E. coli under laboratory 
conditions but whiefc 1 b not on the list 
(Host A), the following type of experi- 
ment may be carried out under PI con- 
ditions without Host A having been 
approved as an HVl host: DNA from 
Host A may be Inserted Into a vector 
and propagated In E. coli K-12 under 
PI conditions. Subsequently, this re- 
combinant DNA may be returned to 
Host A by mobilization, transforma- 
tion. or transduction and may then be 
propagated In Host A In any desired 
vector under PI conditions. 
For a prokaryote which does not ex- 
change genetic Information wfth E. 
coli (Host B). the following type of ex- 
periment may be carried out without 
Host B having been approved as an 
HVl host: DNA from Hoot B may be 
Inserted Into a vector from a certified 
EK2 host-vector system and propagat- 
ed In E. coli K-12 under the appropri- 
ate conta inm e n t conditions tsee sec- 
tion IIl-A-l-b-<2)]. Subsequently, this 
recombinant DNA may be returned to 
Host B and propagated In Host B 
under PI oondltlons.(gj) 
III-C. Experiments with Eukaryotic 
Host- Vector*. 
III-C-1. Vertebrate Host-Vector Sys- 
tem*. 144) (Summary Given In Table 
IV i 
ni-C-l-a. Polyoma Virus. 
III-C- l-a-(l). Productive Virus-Cell 
Interaction*. 
HI-C-l-a-GMa). Defective or Intact 
polyoma virus genomes, with appropri- 
ate helper. If necessary, can be used In 
P2 conditions to propagate DNA se- 
quences: 
III-C-l-a-GMaMI). From bacteria 
of class 1 or class 2(11 or their phages 
or plasmids, except for those that pro- 
duce potent polypeptide toxins; 134) 
III-C-l-a-UMaMZ). Prom mice; 
III-C-l-a-dMaMJ). From eukaryo- 
tic organisms that do not produce 
potent polypeptide toxins. (34) pro- 
vided the DNA segment Is > 99 per- 
cent pure. 
III-C-l-a-GMb). Defective polyoma 
genomes, with appropriate helper. If 
necessary, can be used In P2 conditions 
for shotgun experiments to propagate 
DNA sequences from eukaryotic or- 
FEDHAi lEOISTEk. VOL 43. MO. 144 — FRIDAY, JULY 24. 1971 
[ 41 ] 
