33172 
NOTlCiS 
(1) Viruses a/ Eukaryote*. 
(a) DNA viruses 
1. Nontransf arming viruses. 
(а) Adeno-associated viruses, minute 
virus of mice, and mouse adenoxrirus 
strain FL.— PI physical containment 
Including no mouth pipetting + an 
EK1 host- vector shall be used (or DNA 
recombinants produced with the 
whole genome, subgenomic DNA seg- 
ments. or cDNA copies of cellular 
mRNA.' 
(б) Other viruses 
(I) PI physical containment Includ- 
ing no mouth pipetting + an EK1 
host-vector shall be used (or DNA re- 
combinants produced with purified 
subgenamlc segments or cDNA copies 
of cellular mRNA.* 
(II) PI physical containment Includ- 
ing no mouth pipetting + an EK 1 host 
vector, which. In the case of a plasmid, 
must be non-mobllizable shall be used 
for DNA recombinants produced with 
the whole genome. 
2. Transforming viruses 
(а) Herpes saimiri and herpes ateles 
(I) PI physical containment Includ- 
ing no mouth pipetting + an EK1 host 
vector shall be used for DNA recom- 
binants produced with purified non- 
transforming subgenomic DNA seg- 
ments or cDNA copies of cellular 
mRNA.* 
(II) P2 physical containment + an 
EK1 host vector which. In the case of 
a plasmid, must be non-mobllLzable. 
shall be used for DNA recombinants 
produced with purified subgenomic 
DNA segments containing an entire 
transforming gene. 
(ill) P3 physical containment + an 
EK 1 host-vector or P2 + EK2 shall be 
used for DNA recombinants produced 
with the whole genome. 
(б) Other viruses 
(i) PI physical containment Includ- 
ing no mouth pipetting + an EK 1 
host-vector shall be used for DNA re- 
combinants produced with purified 
non transforming subgenomic DNA 
segments or cDNA copies of cellular 
mRNA.* 
(11) P2 physical containment + an 
EK1 host- vector which. In the case of 
a plasmid, must be non mobillzable, 
shall be used for DNA recombinants 
produced with the whole genome or 
purified subgenomic DNA segments 
containing an entire transforming 
gene. 
(6) RNA viruses 
1. Retro viruses. 
(a) Gibbon ape and woolly monkey 
viruses 
(I) PI physical containment , includ- 
ing no mouth pipetting + an EK1 
host-vector shall be used for DNA re- 
combinants produced with purified 
non-transforming subgenomic DNA 
segments. 
(II) P2 physical containment + an 
EK1 host-vector which, in the case of 
a plasmid, must be non-mobilizable, 
shall be used for DNA recombinants 
produced with purified subgenomic 
DNA segments containing an entire 
transforming gene or cDNA copies of 
cellular mRNA.* 
(Ill) P2 physical containment + an 
EK2 host-vector shall be used for DNA 
recombinants produced with the 
whole genome. 
(b) Other viruses 
(I) PI physical containment Includ- 
ing no mouth pipetting + an EK1 
host-vector shall be used for DNA re- 
combinants produced with purified 
non-transforming subgenomic DNA 
segments. 
(II) P2 physical containment + an 
EK1 host- vector which. In the case of 
a plasmid, must be non-mobllizable, 
shall be used for DNA recombinants 
produced with purified subgenomic 
DNA segments containing an entire 
transforming gene, the whole genome, 
or cDNA copies of cellular mRNA.* 
2. Negative strand viruses— PI phys- 
ical containment Including no mouth 
pipetting + an EK1 host-vector shall 
be used for DNA recombinants pro- 
duced with the whole genome, subgen- 
omic DNa segments or purified cDNA 
copies of cellular mRNA.* 
3. Plus-strand RNA viruses 
(a) Types 1 and 2 Sabin poliovirus 
and strain 17D ( Theiler ) of yellow 
fever trims.— PI physical containment 
Including no mouth ptpetttng + an 
EK1 host- vector shall be used tor DNA 
recombinants produced with the 
whole genome, subgenomic DNA seg- 
ments or purified cDNA copies of cel- 
lular mRNA.* 
(b) Other viruses 
(I) PI physical containment Includ- 
ing no mouth pipetting -f an EK1 
host-vector shall be used for DNA re- 
combinants produced with purified 
subgenomic DNA segments. 
(II) P2 physical containment + an 
EK1 host vector which. In the cse of a 
plasmid, must be nonmobtllzable. shall 
be used for DNA recombinants pro- 
duced with the whole genome or puri- 
fied cDNA copies of cellular mRNA.* 
4 . Double-stranded segmented RNA 
viruses— PI physical containment In- 
cluding no mouth pipetting a- an EK1 
host vector shall be used for DNA re- 
combinants produced with mixtures of 
subgenomic segments, a specific sub- 
genomfc segment, or purified cDNA 
copies of cellular mRNA.* 
5. VXroids— PI physical containment 
including no mouth pipetting + an 
EK1 host-vector shall be used for DNA 
recombinants produced with the 
whole genome, subgenomie DNA seg- 
ments or eDNA copies of cellular 
mRNA.* 
•The cDNA copy of cellular mRNA must 
be 99 percent pure; otherwise, physical and 
biological containment specified for shotgun 
experiments Involving uninfected eukaryo- 
tic cellular DNA (sec. B.l.a(l)] shall be 
used. 
(c) Intracellular viral DNA.— Physi- 
cal and biological containment speci- 
fied for shotgun experiments Involving 
uninfected eukaryotic cellular DNA 
[sec. B.l.a.(l)] shall be used .for DNA 
recombinants produced with Integrat- 
ed viral DNA or viral genomes present 
In Infected cells. 
3. Experiments with Eukaryotic 
host-vectors 
a Vertebrate host- vector systems— 
Because this work will be done almost 
exclusively In tissue culture cells, 
which have no capacity for propaga- 
tion outside the laboratory, the prima- 
ry focus for containment is the vector, 
it should be pointed out that risk of 
laboratory acquired Infection as a con- 
sequence of tissue culture manipula- 
tions Is very low. Given good microbio- 
logical practices, the most likely mode 
of escape of recombinant DNAs from a 
physically contained laboratory Is car- 
riage by an Infected human; thus the 
vector with an Inserted DNA segment 
should have little or no ability to repli- 
cate or spread In humans Further, a 
recombinant virus should not Inad- 
vertently pose a threat to any species. 
For use as a vector In a vertebrate 
host cell system, an animal viral DNA 
molecule should display the following 
properties: 
(a) It should not consist of the whole 
genome of any agent that Is Infectious 
for humans or that replicates to a sig- 
nificant extent in human cells In 
tissue culture. If the recombinant mol- 
ecule is used to transform non-permls- 
slve cells (l.e. cells which do not pro- 
duce Infectious virus particles), this Is 
not a requirement. 
(b) It should be derived from a virus 
whose epidemiological behavior and 
host range are well understood. 
(c) In permissive cells. It should be 
defective when carrying an Inserted 
DNA segment; (Le. propagation of the 
recombinant DNA as a virus must be 
dependent upon the presence of a 
complementing helper genome). In 
almost all cases this condition would 
be achieved automatically by the ma- 
nipulations used to construct and 
propagate the retomblnants. In addi- 
tion. the amount of DNA encapsidated 
In the particles of most animal viruses 
is defined within fairly close limits. 
The Insertion of sizeable foreign DNA 
sequences, therefore, generally de- 
mands a compensatory deletion of 
viral sequences. It may be possible to 
Introduce very short Insertions (50-100 
base pairs) without repdering the viral 
vector defective. In such a situation, 
the requirement that the viral vector 
be defective Is not necessary except In 
those cases In which the Inserted DNA 
encodes a biologically active polypep- 
tide. 
It Is desired but not required that 
the functional anatomy of the vector 
be known— that Is, there should be a 
FSOHAl IWBTlt VOL «J. NO. 14*— KIOAY, JUIY IS. 1971 
[1331 
