59418 
Federal Register / Vol. 46, No. 233 / Friday, December 4. 1981 / Notices 
(having four 8-inch diameter openings) 
without gloves, and (3) with an installed front 
closure panel equipped with arm-length 
rubber gloves, llte face velocity of the 
inward flow of air through the full-width open 
front is 75 feet per minute or greater. A C/osa 
//cabinet is a ventilated cabinet for 
personnel and product protection having an 
open front with Inward air flow for personnel 
protection, and HEPA filtered mass 
recirculated air flow for product protection. 
The cabinet exhaust air is filtered through a 
HEPA Alter. The face velocity of the inward 
flow of air through the full-width open front is 
75 feet per minute or greater. Desi^ and 
performance speciflcations for C/oss // 
cabinets have been adopted by the National 
Sanitation Foundation. Ann Arbor. Michigan. 
A Class III cabinet is a closed-front 
ventilated cabinet of gas-tight construction 
which provides the highest level of personnel 
protection of all biohazard safety cabinets. 
The Interior of the cabinet is protected from 
contaiminants exterior to the cabinet. The 
cabinet is fltted %vith arm-length rubber 
gloves and is operated under a negative 
pressure of at least 0.5 Inches water gauge. 
All supply air is Altered through HEPA Alters. 
Exhaust air is Altered through two HEPA 
Altars or one HEPA Alter and incinerator 
before discharged to the outside environment 
121) HershAeld. V, K W Boyer. C 
Yanofsky. M. A. Lovett and D. R. Helinski 
(1074). Plasmid ColEJ as a Molecular Vehicle 
for Cloning and Amplificabon of DSA. Proc. 
Nat Acad. Sd. USA 71. 3455-3450. 
122) Wensink. P. C. D. ). Flnnegaa ). E. 
Donelson. and D. S. Hogness (1074). A System 
for Mapping DNA Sequences in the 
Chromosomes of Drosophila Melanogaster. 
Call X 315-335. 
(23) Tanaka. T.. and B. Welsblum (1075). 
Construction of a Colicin El-8 Factor 
Composite Plasmid In Vitro: Means for 
Amplification of Deoxyribonucleic Acid. ). 
Bacteriol. 121. 354-382. 
124) Armstrong. K. A.. V. HershAeld. and D. 
R. Helinski (1077). Gene Cloning and 
Containment Properties of Plasmid Col El 
and Its Denratives. Science 190. 172-174. 
(25) Bolivar. F.. R. L Rodriguez. M C 
Betlsch. and H. W. Boyer (1077). Construction 
and Characterisation of Sew Cloning 
Vehicles: I. Ampicillin-Resistant Derivative 
of pMB9 Gene 2 75-03. 
[28) Cohen. S. N.. A. C W. Chang H. Boyer, 
and R. Helling (1073). Construction of 
Biologically Functional Bacteria! Plasmids in 
Vitro. Proc Nall. Acad. Sd. USA 70. 3240- 
3244. 
(27) Bolivar. F.. R. L Rodriquez. R. |. 
Greene. M. C Batlach. H. L Reyneker. H. W. 
Boyer. ). H. Cross, and & Falkow (1077). 
Construction and Characterization of Sew 
Cloning Vehicles: II. A Multi-Purpose 
Cloning System. Gene Z 05-113. 
[28) Thomas. M.. ). R. Cameroa and R. W. 
Davis (1074). Viable Molecular Hybrids of 
Bacteriophage Lambda and Eukaryotic DSA. 
Proc Nat Acad. Sd. USA 71. 4570-4583. 
(20) Murray. N.E.. and K. Murray (1074). 
Manipulation of Restriction Targets in Phage 
Lambda to Form Receptor Chromosomes for 
DSA Fragments. Nature 2S1. 478-481. 
(20) Rambach. A., and P. Tiollais (1074). 
Bacteriophage Having EcoRI Endonuclease 
Sites Only in the Son-Essential Region of the 
Genome. Proc. Nat. Acad. Sci.. USA 71, 3927- 
3930. 
(J/) Blattner. F. R., B. G. Williams. A. E. 
Bleche. K. Denniston-Thompson. H. E. Faber. 
L A. Furlong. D. |. Gunwald. D. O. Kiefer, D. 
0. Moore. ). W. Shumm. E. L Sheldon, and O. 
Smithies (1977). Charon Phages: Safer 
Derivatives of Bacteriophage Lambda for 
DSA Cloning. Sdence 196. 183-169. 
(22) Donohue. D. )., and P. A. Sharp (1977). 
An Improved Lambda Vector Construction of 
Mode! Recombinants Coding for Kanamycin 
Resistance. Gene 1. 209-227. 
(22) Leder, P.. D. Tiemeier and L Enquist 
(1977). EK2 Derivatives of Bacteriophage 
Lambda Useful in the Cloning of DSA from 
Higher Organisms: The gt WES System. 
Science 196. 175-177. 
(23A) Skalka. A. (1978). Current Status of 
Coliphage EK2 Vectors. Geen 2. 29-35. 
(225) Szybalsy. W.. A. Skalka. S. 
Gottesman. A. Campbell, and D. Botstein 
(1078). Standardised Laboratory Tests for 
EK2 Certification. Gene 2. 36-38. 
(24) We are spedAcally concerned with the 
remote possibility that potent toxins could be 
produced by acquiring a single gene or cluster 
of genes. See also footnote 2A. 
(25) DeAned as observable under optimal 
laboratory conditions by transformation, 
transduction, phage Infection, and/or 
conjugation with transfer of phage, plasmid, 
and/or chromosomal genetic information. 
Note that this definition of exchange may be 
less stringent than that applied to exempt 
organisms under Section l-E-4. 
(20) As dassiAed in the Third Report of the 
International Committee on Taxonomy of 
Viruses: GassiAcation and Nomenclature of 
Viruses. R. E. F Matthews. Ed. Intervirology 
12 (129-206) 1070. (As noted in the Prohibition 
Section, the use of viruses classiAed(l) as 
Gass 4 or 5 is prohibited.) 
(27) The cONA copy of the viral mRNA 
must be >90% pure: otherwise as for shotgun 
experiments with eukaryotic cellular DNA. 
[37A) For the purpose of these Guidelines, 
viruses of the families Papovaviridae, 
Adenoviridae, and Herpetoviridae (38) 
should be considered as "transforming” 
viruses. While only certain of these viruses 
have been associated with cell 
transformation in vivo or in vitro, it seems 
prudent to consider all members to be 
potentially capable of transformation. In 
addition, those viruses of the family 
Poxviridae that produce proliferative 
responses — i.e.. myxoma, rabbit and squirrel 
Abroma, and Yaba viruses — should be 
considered as “transforming." 
(20) >00% pure (i.e.. less than 1% of the 
DNA consists of intact viral aenomes): 
otherwise as for whole genomes. 
(29) The viruses have been classiAed by 
NCI as "moderate-risk oncogenic viruses." 
See "Laboratory Safety Monograph — A 
Supplement to the NIH Guidelines for 
Recombinant DNA Research" for 
recommendations on handling the viruses 
themselves. 
[40] (Deleted) 
(4/) The DNA preparation is deAned as 
"purified" if the desired DNA represents at 
least 99% (w/w) of the total DNA in the 
preparation, provided that it was veriAed by 
more than one procedure. 
(42) The lowering of the containment level 
when this degree of puriAcation has been 
obtained is based on the fact that the total 
number of clones that must be examined to 
obtain the desired clone is markedly reduced. 
Thus, the probability of cloning a harmful 
gene could, for example, be reduced by more 
than 10*-fold when a nonrepetitive gene from 
mammals was being sought. Furthermore, the 
level of purity speciAed here makes it easier 
to establish that the desired DNA does not 
contain harmful genes. 
(42) This is not permitted, of course, if it 
fulls under any of the Prohibitions of Section 
1-D. Of particular concern here is prohibition 
l-D-5, i.e., "Deliberate transfer of a drug 
resistance trait to micro-organisms that are 
not known 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." 
(44) Because this work will be done almost 
exclusively in tissue culture cells, which have 
no capacity for propagation outside the 
laboratory, the primary focus for containment 
is the vector. It should be pointed out that 
risk of laboratory-acquired infection as a 
consequence of tissue culture manipulation is 
very low. Given good microbiological 
practices, the most likely mode of escape of 
recombinant DNAs from a physically 
contained laboratory is carriage by an 
Infected human. Thus the vector with an 
inserted DNA segment should have little or 
no ability to replicate or spread in humans. 
For use as a vector in a vertebrate host cell 
system, an animal viral DNA molecule should 
display the following properties; 
(i) It should not consist of the whole 
genome of any agent that is infectious for 
humans or that replicates to a signiAcant 
extent in human cells in tissue culture. If the 
recombinant molecule is used to transform 
nonpermissive cells (i.e., cells which do not 
produce infectious virus particles), this is not 
a requirement. 
(ii) It should be derived from a virus whose 
epidemiological behavior and host range are 
well understood. 
(iii) In permissive cells, it should be 
defective when carrying an inserted DNA 
segment (i.e.. 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 manipulations 
used to construct and propagate the 
recombinants. In addition, the amount of 
DNA encapsidated in the particles of most 
animal viruses is deAned within fairly close 
limits. The insertion of sizable foreign DNA 
sequences, therefore, generally demands a 
compensatory deletion of viral sequences. It 
may be possible to introduce very short 
insertions (50-100 base pairs) without 
rendering 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 polypeptide. 
It is desired but not required that the 
functional anatomy of the vector be known — 
that is. fL-re sLogld be a clear idea of the 
location within the molecule of; 
[ 307 ] 
