APPENDIX I. BIOLOGICAL CONTAINMENT (See Appendix E, Certified Host-Vector Systems)

Appendix I-I. Levels of Biological Containment

In consideration of biological containment, the vector (plasmid, organelle, or virus) for the recombinant DNA and the host (bacterial, plant, or animal cell) in which the vector is propagated in the laboratory will be considered together. Any combination of vector and host which is to provide biological containment shall be chosen or constructed so that the following types of "escape" are minimized: (i) survival of the vector in its host outside the laboratory, and (ii) transmission of the vector from the propagation host to other non-laboratory hosts. The following levels of biological containment (host-vector systems) for prokaryotes are established. Appendices I-I-A through I-II-B describe levels of biological containment (host-vector systems) for prokaryotes. Specific criteria will depend on the organisms to be used.

Appendix I-I-A. Host-Vector 1 Systems

Host-Vector 1 systems provide a moderate level of containment. Specific Host-Vector 1 systems are:

Appendix I-I-A-1. Escherichia coli K-12 Host-Vector 1 Systems (EK1)

The host is always Escherichia coli K-12 or a derivative thereof, and the vectors include non-conjugative plasmids (e.g., pSC101, ColE1, or derivatives thereof (see Appendices I-III-A through G, Footnotes and References of Appendix I) and variants of bacteriophage, such as lambda (see Appendices I-III-H through O, Footnotes and References of Appendix I). The Escherichia coli K-12 hosts shall not contain conjugation-proficient plasmids, whether autonomous or integrated, or generalized transducing phages.

Appendix I-I-A-2. Other Host-Vector 1 Systems

At a minimum, hosts and vectors shall be comparable in containment to Escherichia coli K-12 with a non-conjugative plasmid or bacteriophage vector. Appendix I-II, Certification of Host-Vector Systems, describes the data to be considered and mechanism for approval of Host-Vector 1 systems.

Appendix I-I-B. Host-Vector 2 Systems (EK2)

Host-Vector 2 Systems provide a high level of biological containment as demonstrated by data from suitable tests performed in the laboratory. Escape of the recombinant DNA either via survival of the organisms or via transmission of recombinant DNA to other organisms should be < 1/10⁸ under specified conditions. Specific Host-Vector 2 systems are:

Appendix I-I-B-1. For Escherichia coli K-12 Host-Vector 2 systems (EK2) in which the vector is a plasmid, no more than 1/10⁸ host cells shall perpetuate a cloned DNA fragment under the specified non-permissive laboratory conditions designed to represent the natural environment, either by survival of the original host or as a consequence of transmission of the cloned DNA fragment.

Appendix I-I-B-2. For Escherichia coli K-12 Host-Vector 2 systems (EK2) in which the vector is a phage, no more than 1/10⁸ phage particles shall perpetuate a cloned DNA fragment under the specified non-permissive laboratory conditions designed to represent the natural environment, either as a prophage (in the inserted or plasmid form) in the laboratory host used for phage propagation, or survival in natural environments and transferring a cloned DNA fragment to other hosts (or their resident prophages).

Appendix I-II. Certification of Host-Vector Systems

Appendix I-II-A. Responsibility

Host-Vector 1 systems (other than Escherichia coli K-12) and Host-Vector 2 systems may not be designated as such until they have been certified by the NIH Director. Requests for certification of host-vector systems may be submitted to the Office of Biotechnology Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda, MD 20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839 (fax). Proposed host-vector systems will be reviewed by the RAC (see Section IV-C-1-b-(1)-(f), Major Actions). Initial review will based on the construction, properties, and testing of the proposed host-vector system by a subcommittee composed of one or more RAC members and/or ad hoc experts. The RAC will evaluate the subcommittee's report and any other available information at the next scheduled RAC meeting. The NIH Director is responsible for certification of host-vector systems, following advice of the RAC. Minor modifications to existing host-vector systems (i.e., those that are of minimal or no consequence to the properties relevant to containment) may be certified by the NIH Director without prior RAC review (see Section IV-C-1-b-(2)-(f), Minor Actions). Once a host-vector system has been certified by the NIH Director, a notice of certification will be sent by NIH/OBA to the applicant and to the Institutional Biosafety Committee Chairs. A list of all currently certified host-vector systems is available from the Office of Biotechnology Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda, MD 20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839 (fax). The NIH Director may rescind the certification of a host-vector system (see Section IV-C-1-b-(2)-(g), Minor Actions). If certification is rescinded, NIH will instruct investigators to transfer cloned DNA into a different system or use the clones at a higher level of physical containment level, unless NIH determines that the already constructed clones incorporate adequate biological containment. Certification of an host-vector system does not extend to modifications of either the host or vector component of that system. Such modified systems shall be independently certified by the NIH Director. 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 to a certified host-vector system requires submission of complete testing data.

Appendix I-II-B. Data to be Submitted for Certification

Appendix I-II-B-1. Host-Vector 1 Systems Other than Escherichia 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, survival, and the mechanisms by which it exchanges genetic information; the range of organisms with which this organism normally exchanges genetic information and the type of information is exchanged; and any relevant information about 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 Host-Vector 1 system.

Appendix I-II-B-2. Host-Vector 2 Systems

Investigators planning to request Host-Vector 2 systems certification may obtain instructions from NIH/OBA concerning data to be submitted (see Appendices I-III-N and O, Footnotes and References of Appendix I). 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 non-permissive 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 non-permissive 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 or one or more 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 shall be submitted 12 weeks prior to the RAC meeting at which such data will be considered by the Office of Biotechnology Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda, MD 20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839 (fax). Investigators are encouraged to publish their data on the construction, properties, and testing of proposed Host Vector 2 systems prior to consideration of the system by the RAC and its subcommittee. Specific instructions concerning the submission of data for proposed Escherichia coli K-12 Host-Vector 2 system (EK2) involving either plasmids or bacteriophage in Escherichia coli K-12, are available from the Office of Biotechnology Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda, MD 20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839 (fax).

Appendix I-III. Footnotes and References of Appendix I

Appendix I-III-A. Hersfield, V., H. W. Boyer, C. Yanofsky, M. A. Lovett, and D. R. Helinski, Plasmid Co1E1 as a Molecular Vehicle for Cloning and Amplification of DNA. Proc. Nat. Acad. Sci., 1974, 71, pp. 3455-3459.

Appendix I-III-B. Wensink, P. C., D. J. Finnegan, J. E. Donelson, and D. S. Hogness, A System for Mapping DNA Sequences in the Chromosomes of Drosophila Melanogaster. Cell, 1974, 3, pp. 315-335.

Appendix I-III-C. Tanaka, T., and B. Weisblum, Construction of a Colicin El-R Factor Composite Plasmid in Vitro: Means for Amplification of Deoxyribonucleic Acid. J. Bacteriol., 1975, 121, pp. 354-362.

Appendix I-III-D. Armstrong, K. A., V. Hershfield, and D. R. Helinski, Gene Cloning and Containment Properties of Plasmid Col E1 and Its Derivatives, Science, 1977, 196, pp. 172-174.

Appendix I-III-E. Bolivar, F., R. L. Rodriguez, M. C. Betlack, and H. W. Boyer, Construction and Characterization of New Cloning Vehicles: I. Ampicillin-Resistant Derivative of PMB9, Gene, 1977, 2, pp. 75-93.

Appendix I-III-F. Cohen, S. N., A. C. W. Chang, H. Boyer, and R. Helling. Construction of Biologically Functional Bacterial Plasmids in Vitro. Proc. Natl. Acad, Sci., 1973, 70, pp. 3240-3244.

Appendix I-III-G. Bolivar, F., R. L. Rodriguez, R. J. Greene, M. C.Batlack, H. L. Reyneker, H. W. Boyer, J. H. Cross, and S. Falkow, 1977, Construction and Characterization of New Cloning Vehicles II. A Multi-Purpose Cloning System, Gene, 1977, 2, pp. 95-113.

Appendix I-III-H. Thomas, M., J. R. Cameron, and R. W. Davis (1974). Viable Molecular Hybrids of Bacteriophage Lambda and Eukaryotic DNA. Proc. Nat. Acad. Sci., 1974, 71, pp. 4579-4583.

Appendix I-III-I. Murray, N. E., and K. Murray, Manipulation of Restriction Targets in Phage Lambda to Form Receptor Chromosomes for DNA Fragments. Nature, 1974, 51, pp. 476-481.

Appendix I-III-J. Ramback, A., and P. Tiollais (1974). Bacteriophage Having EcoRI Endonuclease Sites Only in the Non-Essential Region of the Genome. Proc. Nat. Acad. Sci., 1974, 71, pp. 3927-3820.

Appendix I-III-K. Blattner, F. R., B. G. Williams, A. E. Bleche, K. Denniston-Thompson, H. E. Faber, L. A. Furlong, D. J. Gunwald, D. O. Kiefer, D. D. Moore, J. W. Shumm, E. L. Sheldon, and O. Smithies, Charon Phages: Safer Derivatives of Bacteriophage Lambda for DNA Cloning, Science 1977, 196, pp. 163-169.

Appendix I-III-L. Donoghue, D. J., and P. A. Sharp, An Improved Lambda Vector: Construction of Model Recombinants Coding for Kanamycin Resistance, Gene, 1977, 1, pp. 209-227.

Appendix I-III-M. Leder, P., D. Tiemeier and L. Enquist (1977), EK2 Derivatives of Bacteriophage Lambda Useful in the Cloning of DNA from Higher Organisms: The λgt WES System, Science, 1977, 196, pp. 175-177.

Appendix I-III-N. Skalka, A., Current Status of Coliphage AEK2 Vectors, Gene, 1978, 3, pp. 29-35.

Appendix I-III-O. Szybalski, W., A. Skalka, S. Gottesman, A. Campbell, and D. Botstein, Standardized Laboratory Tests for EK2 Certification, Gene, 1978, 3, pp. 36-38.