77394 
Federal Register / Vol. 45, No. 227 / Friday, November 21, 1980 / Notices 
Table III .—Recommended Containment for Cloning of Viral DNA or cDNA in Certain HV1 and HV2 Systems 
Specified in Appendix O — Continued 
[See text lor lull details) 
Type of viral DNA segment to be cloned 
Subgenomic [38] Genomic' cDNA 
, from viral 
mRNA (37) 
Virus class Non- Segment Nonsegmented Segmented 
transforming containing genome genome 
segment an entire 
transforming 
gene 
Other P1+HVU38] 
RNA: 
Retroviruses: 
Gibbon ape, wooly monkey FeLV and P1+HVK38] 
FeSV [39], 
Other P1+HVU38] 
Negative-Strand RNA P1+HV1 
Plus-Strand RNA: 
Types 1 and 2 Sabin polio, 17D yellow fever P1+HV1 
vaccine strains. 
Other PI + HVl [38] 
Double-stranded RNA P1+HV1 
Plant viruses +viroids P1+HV1 
Intracellular viral DNA 2 
P2 + HV1 P2 + HV1 P2 + HV1 
P2 + HV1 P2 + HV2 P2 + HV2 
or P3 + HV1 or P3 + HV1 
P2+HV1 P2+HV1 P2 + HV1 
P1+HV1 P1+HV1 P1+HV1 
P1+HV1 P1+HV1 
P2 + HV1 P2 + HV1 
P1+HV1 P1+HV1 
PI + HVl P1+HV1 P1+HV1 
2 2 
' See exception given at asterisk at end of appendix D. 
2 See text. 
III-A-2-a-(2)-(c)-(2)-(Zj). P2 physical 
containment + an HVl 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 .(37) 
III— A— 2— a— (2)— (d). Double-Stranded 
Segmented RNA Viruses. Pi physical 
containment + 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/37; 
III-A-2-a-(2)-(e). RNA Plant Viruses 
and Plant Viroids. [48) Pll physical 
containment -f 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) purified cDNA 
copies of viral mRNA/37y 
III-A-2-a-(3). Intracellular Viral 
DNA. Physical and biological 
containment specified for shotgun 
experiments with eukaryotic cellular 
DNA [see Section III— A— (1)— (a)] shall be 
used for DNA recombinants produced 
with integrated viral DNA or viral 
genomes present in infected cells. 
III-A-2-b. Eukaryotic Organelle 
DNAs. P2 phycial containment + an 
HVl host-vector, or Pll + 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 III— A— 
1-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 (5) can be carried out under 
lower containment conditions than used 
for the corresponding shotgun 
experiment.^?). The containment may 
be decreased one step in physical 
containment (P4 + P3; P3 + P2; P2 + P1) 
while maintaining the biological 
containment specified for the shotgun 
experiment, or one step in biological 
containment (HV3 + HV2; HV2 + HV1) 
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-a to 
levels below PI + HVl, which requires 
prior NIH approval. (See Section IV-E- 
1- 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 
(3) 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 -|- 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).) 
Ill— B. Experiments with Prokaryotic 
Host- Vectors Other Than E. coli K-12 
III— 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 III-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. 
Ill— B— 2. Return of DNA Segments to 
Prokaryotic Non-HVl Host of Origin. 
Certain experiments involving those 
prokaryotes that echange 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^) 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 under Pi 
conditions. 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 under Pi conditions. Subsequently, 
this recombinant DNA may be returned 
to Host B and progagated in Host B 
under PI conditions. [43) 
[13] 
