Federal Register / Vol. 49, No. 227 / Friday, November 23, 1984 / Notices 
46287 
of transmission of the cloned DNA 
fragment. 
Appendix I-l-B-2. For EK2 host- 
vector systems in which the vector is a 
phage, no more than one in 10* phage 
particles should be able to perpetuate a 
cloned DMA fragment under the 
specified nonpermissive laboratory 
conditions designed to represent the 
natural environment either: (it) as a 
prophage (in the inserted or plasmid 
form) in the laboratory host used for 
phage propagation or (ii) by surviving in 
natural environments and transferring a 
cloned DNA fragment to other hosts (or 
their resident prophages). 
Appendix /-//. Certification of Host- 
Vector Systems. 
Appendix I-II-A. Responsibility. HVl 
systems other than E. coli K-12 and HV2 
host-vector systems may not be 
designated as such until they have been 
certified by the Director, NIH. 
Application for certification of a host- 
vector system is made by written 
application to the Office of Recombinant 
DNA Activities, National Institutes of 
Health, Building 31, Room 3B10, 
Bethesda, Maryland 20205. 
Host-vector systems that are proposed 
for certification will be reviewed by the 
RAC (see Section I V— C— 1— b— (1) — (e)). 
This will first involve review of the data 
on construction, properties, and testing 
of the proposed host-vector system by a 
working group composed on one or more 
members of the RAC and other persons 
chosen because of their expertise in 
evaluating such data. The committee 
will then evaluate the report of the 
working group and any other available 
information at a regular meeting. The 
Director, NIH, is responsible for 
certification after receiving the advice of 
the RAC. Minor modifications of 
existing certified Host-vector systems 
where the modifications are of minimal 
or no consequence to the properties 
relevant to containment may be certified 
by the Director, NIH, without review by 
the RAC (see Section I V— C— 1— b — (3)— (c)). 
When new host-vector systems are 
certified, notice of the certification will 
be sent by ORDA to the applicant and to 
all IBCs and will be published in the 
Recombinant DNA Technical Bulletin. 
Copies of a list of all currently certified 
host-vector systems may be obtained 
from ORDA at any time. 
The Director, NIH, may at any time 
rescind the certification of any host- 
vector system (see Section IV-C-l-b- 
(3)-(d)). If certification of a host-vector 
system is rescinded, NIH will instruct 
investigators to transfer cloned DNA 
into a different system or use the clones 
at a higher physical containment level 
unless NIH determines that the already 
constructed clones incorporate adequate 
biological containment. 
Certification of a given system does 
not extend to modifications of either the 
host or vector component of that system. 
Such modified systems must be 
independently certified by the Director, 
NIH. If modifications are minor, it may 
only be necessary for the investigator to 
submit data showing that the 
modifications have either improved or 
not impaired the major phenotypic traits 
on which the containment of the system 
depends. Substantial modifications of a 
certified system require the submission 
of complete testing data. 
Appendix I-Il-B. Data to be 
Submitted for Certification. 
Appendix 1-I1-B-1. HVl Systems 
Other than E. coli K-12. The following 
types of data shall be submitted, 
modified as appropriate for the 
particular system under consideration: 
(i) a description of the organism and 
vector; the strain’s natural habitat and 
growth requirements; its physiological 
properties, particularly those related to 
its reproduction and survival and the 
mechanisms by which it exchanges 
genetic information; the range of 
organisms with which this organism 
normally exchanges genetic information 
and what sort of information is 
exchanged; and any relevant 
information on its pathogenicity or 
toxicity; (ii) a description of the history 
of the particular strains and vectors to 
be used, including data on any 
mutations which render this organism 
less able to survive or transmit genetic 
information; and (iii) a general 
description of the range of experiments 
contemplated with emphasis on the 
need for developing such an HVl 
system. 
Appendix I-lI-B-2. HV2 Systems. 
Investigators planning to request HV2 
certification for host-vector systems can 
obtain instructions from ORDA 
concerning data to be submitted(74-15). 
In general, the following types of data 
are required: (i) description of 
construction steps with indication of 
source, properties, and manner of 
introduction of genetic traits; (ii) 
quantitative data on the stability of 
genetic traits that contribute to the 
containment of the system; (iii) data on 
the survival of the host-vector system 
under nonpermissive laboratory 
conditions designed to represent the 
relevant natural environment; (iv) Data 
on transmissibility of the vector and/or 
a cloned DNA fragment under both 
permissive and nonpermissive 
conditions; (v) data on all other 
properties of the system which affect 
containment and utility, including 
information on yields of phage or 
plasmid molecules, ease of DNA 
isolation, and ease of transfection or 
transformation; and (vi) in some cases, 
the investigator may be asked to submit 
data on survival and vector 
transmissibility from experiments in 
which the host-vector is fed to 
laboratory animals and human subjects. 
Such in vivo data may be required to 
confirm the validity of predicting in vivo 
survival on the basis of in vitro 
experiments. 
Data must be submitted in writing to 
ORDA. Ten to twelve weeks are 
normally required for review and 
circulation of the data prior to the 
meeting at which such data can be 
considered by the RAC. Investigators 
are encouraged to publish their data on 
the construction, properties, and testing 
of proposed HV2 systems prior to 
consideration of the system by the RAC 
and its subcommittee. More specific 
instructions concerning the type of data 
to be submitted to NIH for proposed EK2 
systems involving either plasmids or 
bacteriophage in E. coli K-12 are 
available from ORDA. 
Appendix I— III — Footnotes and References of 
Appendix I 
(1) Hershfield, V., H. W. Boyer, C. 
Yanofsky, M. A. Lovett, and D. R. Helinski 
(1974). Plasmid CoIEl as a Molecular Vehicle 
for Cloning and Amplification of DNA. Proc. 
Nat. Acad. Sci. USA 71. 3455-3459. 
( 2 ) Wensink, P. C., D. J. Finnegan, J. E. 
Donelson, and D. S. Hogness (1974). A System 
for Mapping DNA Sequences in the 
Chromosomes of Drosophila Melanogaster. 
Cell 3, 315-335. 
(,i) Tanaka, T„ and B. Weisblum (1975). 
Construction of a Colicin El-R Factor 
Composite Plasmid In Vitro: Means for 
Amplification of Deoxyribonucleic Acid. J. 
Bacteriol. 121. 354-362. 
(4) Armstrong, K. A., V. Hershfield, and D. 
R. Helinski (1977). Gene Cloning and 
Containment Properties of Plasmid Col El 
and Its Derivatives, Science 196, 172-174. 
(5) Bolivar, F„ R. L. Rodriguez, M. C. 
Betlach, and H. W. Boyer (1977). Construction 
and Characterization of New Cloning 
Vehicles: I. Ampicillin-Resistant Derivative 
of pMB9. Gene 2, 75-93. 
(6) Cohen, S. N., A. C. W. Chang, H. Boyer, 
and R. Helling (1973). Construction of 
Biologically Functional Bacterial Plasmids in 
Vitro. Proc. Natl. Acad. Sci. USA 70 3240- 
3244. 
(7) Bolivar, F., R. L. Rodriguez, R. J. Greene, 
M. C. Batlach, H. L. Reyneker, H. W. Boyer, J. 
H. Crosa, and S. Falkow (1977). Construction 
and Characterization of New Cloning 
Vehicles: II. A Multi-Purpose Cloning 
System. Gene 2. 95-113. 
(8) Thomas, M., J. R. Cameron, and R. W. 
Davis (1974). Viable Molecular Hybrids of 
Bacteriophage Lambda and Eukaryotic DNA. 
Proc. Nat. Acad. Sci. USA 71. 4579-4583. 
(9) Murruy, N. E., and K. Murray (1974). 
Manipulation of Restriction Targets in Phage 
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