Exemptions and Host Vector Systems Guide
Guide to the NIH Guidelines for Recombinant DNA Research
Exemptions and Host-Vector Systems Guide
|APPENDIX A.||EXEMPTIONS UNDER SECTION III-F-5--SUBLISTS OF NATURAL EXCHANGERS|
Certain specified recombinant DNA molecules that consist entirely of DNA segments from different species that exchange DNA by known physiological processes, though one or more of the segments may be a synthetic equivalent are exempt from these NIH Guidelines (see Section III-F-5, Exempt Experiments). Institutional Biosafety Committee registration is required by MSSM (not required by the NIH) for these exempt experiments. A list of such exchangers will be prepared and periodically revised by the NIH Director with advice from the RAC after appropriate notice and opportunity for public comment (see Section IV-C-1-b-(1)-(c), NIH Director--Specific Responsibilities). For a list of natural exchangers that are exempt from the NIH Guidelines, see Appendices A-I through A-VI, Exemptions Under Section III-F-5 Sublists of Natural Exchangers. Section III-F-5, Exempt Experiments, describes recombinant DNA molecules that are: (1) composed entirely of DNA segments from one or more of the organisms within a sublist, and (2) to be propagated in any of the organisms within a sublist (see Classification of Bergey's Manual of Determinative Bacteriology; 8th edition, R. E. Buchanan and N. E. Gibbons, editors, Williams and Wilkins Company; Baltimore, Maryland 1984). Although these experiments are exempt, it is recommended that they be performed at the appropriate biosafety level for the host or recombinant organism (see *Biosafety in Microbiological and Biomedical Laboratories, 3rd edition, May 1993, U.S. DHHS, Public Health Service, Centers for Disease Control and Prevention, Atlanta, Georgia, and NIH Office of Biosafety, Bethesda, Maryland).
Appendix A-I. Sublist A
Genus Salmonella - including Arizona
Genus Citrobacter - including Levinea
Genus Klebsiella - including oxytoca
Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, and Pseudomonas mendocina
Appendix A-II. Sublist B
Appendix A-III. Sublist C
Appendix A-IV. Sublist D
Appendix A-V. Sublist E
One way transfer of Streptococcus mutans or Streptococcus lactis DNA into Streptococcus sanguis
Appendix A-VI. Sublist F
APPENDIX E. CERTIFIED HOST-VECTOR SYSTEMS
While many experiments using Escherichia coli K-12, Saccharomyces cerevisiae, and Bacillus subtilis are currently exempt from the NIH Guidelines under Section III-F, Exempt Experiments, some derivatives of these host-vector systems were previously classified as Host-Vector 1 Systems or Host-Vector 2 Systems. A listing of those systems follows:
Appendix E-I. Bacillus subtilis
Appendix E-I-A. Bacillus subtilis Host-Vector 1 Systems
The following plasmids are accepted as the vector components of certified B. subtilis systems: pUB110, pC194, pS194, pSA2100, pE194, pT127, pUB112, pC221, pC223, and pAB124. B. subtilis strains RUB 331 and BGSC 1S53 have been certified as the host component of Host-Vector 1 systems based on these plasmids.
Appendix E-I-B. Bacillus subtilis Host-Vector 2 Systems
The asporogenic mutant derivative of Bacillus subtilis, ASB 298, with the following plasmids as the vector component: pUB110, pC194, pS194, pSA2100, pE194, pT127, pUB112, pC221, pC223, and pAB124.
Appendix E-II. Saccharomyces cerevisiae
Appendix E-II-A. Saccharomyces cerevisiae Host-Vector 2 Systems
The following sterile strains of Saccharomyces cerevisiae, all of which have the ste-VC9 mutation, SHY1, SHY2, SHY3, and SHY4. The following plasmids are certified for use: YIp1, YEp2, YEp4, YIp5, YEp6, YRp7, YEp20, YEp21, YEp24, YIp25, YIp26, YIp27, YIp28, YIp29, YIp30, YIp31, YIp32, and YIp33.
Appendix E-III. Escherichia coli
Appendix E-III-A. Escherichia coli (EK2) Plasmid Systems
The Escherichia coli K-12 strain chi-1776. The following plasmids are certified for use: pSC101, pMB9, pBR313, pBR322, pDH24, pBR325, pBR327, pGL101, and pHB1. The following Escherichia coli/S. cerevisiae hybrid plasmids are certified as EK2 vectors when used in Escherichia coli chi-1776 or in the sterile yeast strains, SHY1, SHY2, SHY3, and SHY4: YIpI, YEp2, YEp4, YIp5, YEp6, YRp7, YEp20, YEp21, YEP24, YIp25, YIp26, YIp27, YIp28, YIp29, YIp30, YIp31, YIp32, and YIp33.
Appendix E-III-B. Escherichia coli (EK2) Bacteriophage Systems
The following are certified EK2 systems based on bacteriophage lambda:
|8gt ZJ vir8B'||Escherichia coli K-12|
|Charon 3A||DP50 or DP50supF|
|Charon 4A||DP50 or DP50supF|
|Charon 16A||DP50 or DP50supF|
|Charon 23A||DP50 or DP50supF|
|Charon 24A||DP50 or DP50supF|
Escherichia coli K-12 strains chi-2447 and chi-2281 are certified for use with lambda vectors that are certified for use with strain DP50 or DP50supF provided that the su-strain not be used as a propagation host.
Appendix E-IV. Neurospora crassa
Appendix E-IV-A. Neurospora crassa Host-Vector 1 Systems
The following specified strains of Neurospora crassa which have been modified to prevent aerial dispersion:
In1 (inositolless) strains 37102, 37401, 46316, 64001, and 89601. Csp-1 strain UCLA37 and csp-2 strains FS 590, UCLA101 (these are conidial separation mutants).
Eas strain UCLA191 (an "easily wettable" mutant).
Appendix E-V. Streptomyces
Appendix E-V-A. Streptomyces Host-Vector 1 Systems
The following Streptomyces species: Streptomyces coelicolor, S. lividans, S. parvulus, and S. griseus. The following are accepted as vector components of certified Streptomyces Host-Vector 1 systems: Streptomyces plasmids SCP2, SLP1.2, pIJ101, actinophage phi C31, and their derivatives.
Appendix E-VI. Pseudomonas putida
Appendix E-VI-A. Pseudomonas putida Host-Vector 1 Systems
Pseudomonas putida strains KT2440 with plasmid vectors pKT262, pKT263, and pKT264.
APPENDIX I. BIOLOGICAL CONTAINMENT
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, Co1E1, or derivatives thereof 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
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. 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 8gt 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.