34472 
Federal Register / Vol. 46, No. 126 / Wednesday. July 1, 1981 / Notices 
T*M« WX.— Recommended Containment for Cloning of Viral DNA or cDNA in Certain HV1 and 
HV2 Systems Specified in Appendix O— Continued 
[S«e t«xl lor full details] 
Type of viral ONA segment to be cloned 
Subganomfc (S0) 
Genomic ' 
virus Clast 
Nontransforming 
segment 
Soomont 
contairdrig an 
entire 
trantfomWog 
gene 
Nonsegmented 
genome 
Segmented 
genome 
cDNA from viral 
mRNA [37] 
Ooubte-Slrandad RNA 
Plant VIrutes+VIrolds. 
PI+HV1 
PI -f HVl 
PI +HV1 
PI +HV1 
PI +HV1 
... Pl-t-HVI, 
... P1+HVt 
Intracellular Viral DNA 
■ See exception given at aateriak at and of Appendix D. 
ni-A-2-a-{2HcH2H^)- P2 physical 
containment + an PWl host-vector shall 
be used for DNA recombinants 
produced with (i] cDNA copies of the 
whole genome, or (ii) purified cDNA 
copies of viral mRNA. (57) 
III-A-2-a-(2)-{d). Double-Stranded 
Segmented RNA Viruses. PI physical 
containment -t- an HVl host-vector shall 
be used for DNA recombinants 
produced with (i) mixtures of 
subgenomic cDNA segments, (ii) a 
specific subgenomic cDNA segment, or 
(iii) purified cDNA copies of viral 
mRNA. (57) 
in-A-2-a-^2)-(e). RNA Plant Viruses 
and Plant Viroids. [4B) PI physical 
containment an HVl host-vector shall 
be used for DNA recombinants 
produced with (i) cDNA copies of the 
whole viral genome, (ii) subgenomic 
cDNA segments, or (iii) puriHed cDNA 
copies of viral mRNA. (57) 
ni-A-2-a-(3). Intracellular Viral 
DNA. Physical and biological 
containment specifled for shotgun 
experiments with eukaryotic cellular 
DNA [see Section IIl-A-(l)-(a)] shall be 
used for DNA recombinants produced 
with integrated viral DNA or viral 
genomes present in infected cells. 
III-A-2-a-b. Eukaryotic Organelle 
DNAs. P2 physical containment -|- an 
HVl host-vector, or P1-I-HV2, for 
mitochondrial or chloroplast DNA from 
eukaryotes when the organelle DNA has 
been obtained from isolated organelles. 
Otherwise, the conditions given for 
shotgun experiments apply. 
III-A-2-C. Prokaryotic Plasmid and 
Phage DNAs. The containment levels 
required for shotgun experiments with 
DNA from prokaryotes apply to their 
plasmids or phages (See Section 
UI-A-l-b.) 
III-A-3. Lowering of Containment 
Levels for Characterized or Purified 
DNA Preparations and Clones. Many of 
the risks which might conceivably arise 
from some types of recombinant DNA 
experiments, particularly shotgun 
experiments, would result from the 
inadvertent cloning of a harmful 
sequence. Therefore, in cases where the 
risk of inadvertently cloning the 
“wrong” DNA is reduced by prior 
enrichment for the desired piece, or in 
which a clone made from a random 
assortment of DNAs has been purified 
and the absence of harmful sequences 
established, the containment conditions 
for further work may be reduced. The 
following section outlines the 
mechanisms for such reductions. 
III-A-3-a. Purified DNA Other than 
Plasmids, Bacteriophages, and Other 
Viruses. The formation of DNA 
recombinants from cellular DNAs that 
have been purified [41] and in which the 
absence of harmful sequences has been 
established [3] can be carried out under 
lower containment conditions than used 
for the corresponding shotgun 
experiment. [42] The containment may 
be decreased one step in physical 
containment (P4, P3; P3, P2; P2, Pi) while 
maintaining the biological containment 
specified for the shotgun experiment, or 
one step in biological containment (HV3; 
HV2; HV2; HVl) while maintaining the 
specified physical containment. The 
Institutional Biosafety Committee (IBC) 
must review such a reduction and the 
approval of the IBC and of the NIH must 
be secured before such a reduction may 
be put into effect. IBC approval is 
sufficient for such a reduction except for 
any lowering of containment under 
Section III-A-3 to levels below 
Pi -I- HVl, which requires prior NIH 
approval. (See Section IV-E-l-b-(3)- 
(e).) 
III-A-3-b. Characterized Clones of 
DNA Recombinants. When a cloned 
DNA recombinant has been rigorously 
characterized and the absence of 
harmful sequences has been established 
(5), experiments involving this 
recombinant DNA may be carried out 
under lower containment conditions. 
Institutional Biosafety Committees 
(IBCs) may 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 harmful genes. IBC 
approval is sufficient for such a 
reduction except for any lowering of 
containment under Section III-A-3-b to 
levels below Pi -i- HVl, or reduction of 
containment levels by more than one 
step, which also requires prior NIH 
approval. (See Section 
IV-E-l-b-3-(e).) 
IIl-B. Experiments with Prokaryotic 
Host- Vectors Other Than E. coli K-12. 
IIl-B-1. HVl and HV2 Systems. 
Certain certified HVl and HV2 host- 
vector systems appear in Appendix D. 
The containment levels for these 
systems are given in the subsections of 
Section IIl-A. Other systems in the 
future may be certified as HVl and HV2. 
At the time of certification, the 
classification of containment levels for 
experiments using them will be assigned 
by NIH. 
III-B-2. Return of DNA Segments to 
Prokaryotic Non-HVl Host of Origin. 
Certain experiments involving those 
prokaryotes that exchange genetic 
information with E coli by known 
physiological processes will be exempt 
from these Guidelines if they appear on 
the “list of exchangers" set forth in 
Appendix A (see Section I-E-4). For a 
prokaryote which can exchange genetic 
information [35] with E. coli under 
laboratory conditions but which is not 
on the list (Host A), the following type of 
experiment may be carried out under Pi 
conditions 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. 
Subsequently, this recombinant DNA 
may be returned to Host A by 
mobilization, transformation, or 
transduction and may then be 
propagated in Host A in any desired 
vector under Pi conditions. 
For a prokaryote which does not 
exchange genetic information with E. 
coli (Host B), the following type of 
experiment may be carried out without 
Host B having been approved as an HVl 
host: DNA from Host B may be inserted 
into a vector and propagated in E. coli 
K-12. Subsequently, this recombinant 
DNA may be returned to Host B and 
propagated in Host B under Pi 
conditions. [43) 
III-B-3. Non-HVl Systems. 
Containment levels for other classes of 
experiments involving non-HVl systems 
may be approved by the Director, NIH. 
(See Sections IV-E-l-b-(l)-(b). IV-E-1- 
b-(2)-(c), and IV-E-l-b-(3)-(b).y 
III-C. Experiments with Eukaryotic 
Host-Vectors. 
III-C-1. Vertebrate Host-Vector 
Systems. (44) The subsections of 
Sections III-C-1 -a, -b, -c and -d 
involve the use of specific viral vectors, 
namely polyoma, SV40, human 
adenoviruses 2 and 5, and mouse 
adenovirus strain FL, respectively. The 
[160] 
