NIH Guidelines for rDNA

Compliance with the NIH Guidelines for Research Involving Recombinant DNA Molecules is mandatory for every institution that receives NIH funding for research involving recombinant DNA (rDNA).  It is the responsibility of the Principal Investigator (PI) to make sure that his/her laboratory is in compliance.

Here are links to the official NIH pages:

HTML: https://osp.od.nih.gov/wp-content/uploads/2019_NIH_Guidelines.htm

PDF: https://osp.od.nih.gov/wp-content/uploads/NIH_Guidelines.pdf

The following is a summary of experiments covered by the NIH Guidelines and is intended to assist you in determining which category/categories your experiments fall under.  It may not necessarily be edited for updates made to the official documents linked above.

Section III-A
Experiments Requiring IBC Approval, Recombinant DNA Advisory Committee (RAC) Review, and NIH Director Approval Before Initiation

III-A-1-a.  This category is limited to studies that involve the deliberate transfer of a drug resistance trait to microorganisms that are not known to acquire the trait naturally if such acquisition could compromise the use of the drug to control disease agents in humans, veterinary medicine, or agriculture (antibiotic resistance markers used for selecting and propagating plasmids in E. coli are not included).

Examples:  Cloning a gene for rifampin resistance into Mycobacterium tuberculosis, cloning a gene for tetracycline resistance into Chlamydia trachomatis

Section III-B
Experiments Requiring IBC and NIH/OBA Approval Before Initiation

III-B-1.  This category is limited to experiments involving the cloning of toxin molecules with LD50 of < 100 ng/kg body weight

Examples: Botulinum toxins, tetanus toxin, diphtheria toxin, Shigella dysenteriae neurotoxin

Section III-C
Experiments Requiring IBC and IRB Approval and RAC Review Before Initiation

III-C-1.  This category pertains to experiments involving the deliberate transfer of rDNA, or DNA or RNA derived from rDNA, into one or more human research participants (human gene therapy).  All requirements of Appendix M apply to human gene therapy studies.

Sections III-D, III-E, and III-F

The majority of the rDNA research at UF falls into one of these sections.  The information regarding the various applications in these categories is presented in table format on the following pages for quick reference.

Section III-D
Experiments Requiring IBC Approval Before Initiation

Section Subsection Research Application Description Comments
III-D-1 Experiments using Risk Group (RG) 2, 3, or 4, or Restricted Agents as host-vector systems USDA/APHIS permit may be required for work with certain plant or animal pathogens.  See http://www.aphis.usda.gov/.
III-D-1-a rDNA into RG2 agents Usually conducted at BSL-2/ABSL-2
III-D-1-b rDNA into RG3 agents Usually conducted at BSL-3/ABSL-3
III-D-1-c rDNA into RG4 agents Usually conducted at BSL-4/ABSL-4[1]
III-D-1-d rDNA into restricted agents Containment determined on a case-by-case basis following NIH/OBA review
III-D-2  Experiments in which DNA from Risk Group 2, 3, or 4, or Restricted Agents is cloned into nonpathogenic prokaryotic or lower eukaryotic host-vector systems
III-D-2-a rDNA from RG2, RG3, or RG4 agents BSL-2 containment for cloning from RG2 or RG3 pathogens; specific lowering to BSL-1 may be approved by the IBC

BSL-2 for cloning from RG4 agents only after demonstration that a totally & irreversibly defective fragment of the agent’s genome is present in a given recombinant; otherwise BSL-4 required.
III-D-2-b rDNA from restricted agents Containment determined on a case-by-case basis following NIH/OBA review
III-D-3 Experiments involving the use of infectious DNA or RNA viruses or defective DNA or RNA viruses in the presence of helper virus in tissue culture systems
III-D-3-a Infectious/defective RG2 viruses with helper virus Usually conducted at BSL-2
III-D-3-b Infectious/defective RG3 viruses with helper virus Usually conducted at BSL-3
III-D-3-c Infectious/defective RG4 viruses with helper virus Usually conducted at BSL-4[1]
III-D-3-d Infectious/defective restricted poxviruses with helper virus Containment determined on a case-by-case basis following NIH/OBA review
III-D-3-e Viruses not covered in III-D-3-a through III-D-3-d Usually conducted at BSL-1
III-D-4 Experiments involving whole animals in which the genome has been altered by stable introduction into the germ-line (transgenic animals) and experiments involving viable rDNA-modified microorganisms tested on whole animals
III-D-4-a rDNA from any source except for > 2/3 of eukaryotic viral genome Usually conducted at BSL-1; viral vectors must not lead to transmissible infection
III-D-4-b rDNA involving whole animals and not covered by III-D-1 or III-D-4-a Appropriate containment decided by the IBC
III-D-4-c-(1) Generating transgenic rodents that require BSL-1 containment Covered under Section III-E-3
III-D-4-c-(2) Purchase or transfer of transgenic rodents Exempt under Section III-F-6, Appendix C-VI
III-D-5 Experiments involving whole plants – genetically engineering plants by rDNA methods, using or propagating such plants, using plants with microorganisms or insects containing rDNA
III-D-5-a Exotic plant pathogens with recognized potential for serious detrimental impact on ecosystems Usually conducted at BSL-2+P/BSL-3P
III-D-5-b Readily transmissible exotic agents in which the complete and functional genome may be reconstituted in planta Usually conducted at BSL-2+P/BSL-3P
III-D-5-c Readily transmissible exotic agents in the presence of their arthropod vector Usually conducted at BSL-4P[1]
III-D-5-d Sequences encoding potent vertebrate toxins introduced into plants or associated organisms Usually conducted at BSL-3P
III-D-5-e Microbial pathogens of insects or small animals associated with plants Usually conducted at BSL-2+P/BSL-3P
III-D-6 Experiments involving > 10 liters of culture Containment to be decided by IBC; see Appendix K for containment conditions
III-D-7 Experiments involving influenza viruses Conducted at the containment level corresponding to the RG of the virus that is the source of the majority of segments
III-D-7-a Human H2N2 (1957-1968) BSL-3+ for viruses containing H2 hemagglutinin (HA) segment

BSL-2 for H2 HA gene in cold-adapted, live attenuated vaccine strains and for H2N2 genes other than HA
III-D-7-b Highly pathogenic avian influenza H5N1 Usually conducted at BSL-3+
III-D-7-c 1918 H1N1 Usually conducted at BSL-3+
III-D-7-d Antiviral susceptibility for genes from viruses in III-D-7-a through III-D-7-c Higher containment may be required if any of the genes are resistant to both classes of current antiviral agents (adamantanes and neuraminidase inhibitors)

[1]BSL-4 agents cannot be used at UF

Section III-E
Experiments Requiring IBC Registration and Notice Simultaneous with Initiation

Section Subsection Research Application Description Comments
III-E All components derived from non-pathogenic prokaryotes and non-pathogenic lower eukaryotes BSL-1
III-E-1 Formation of rDNA molecules containing no more than 2/3 of the genome of any eukaryotic virus BSL-1, provided that cells lack helper virus for the specific families of defective virus being used
III-E-2 rDNA-modified plants or rDNA-modified microorganisms associated with plants not covered in sections III-A, III-B, III-D or III-F
III-E-2-a rDNA-modified plants that are not noxious weeds and rDNA-modified non-exotic microorganisms (e.g. Rhizobium and Agrobacterium spp.) BSL-1P
III-E-2-b-(1) Noxious weeds or can interbreed with noxious weeds in the immediate area BSL-1+P/BSL-2P
III-E-2-b-(2) Plants containing complete genome of non-exotic infectious agent BSL-1+P/BSL-2P
III-E-2-b-(3) Plants associated with rDNA-modified non-exotic microorganisms that have potential for serious detrimental impact on ecosystems BSL-1+P/BSL-2P
III-E-2-b-(4) Plants associated with rDNA-modified non-exotic microorganisms that have no potential for serious detrimental impact on ecosystems BSL-1+P/BSL-2P
III-E-2-b-(5) rDNA-modified arthropods or small animals associated with plants or arthropods or small animals with rDNA-modified microorganisms associated with them if the microorganism has no potential for serious detrimental impact on ecosystems BSL-1+P/BSL-2P
III-E-3 Creation of transgenic rodents BSL-1; experiments requiring BSL-2 or higher covered under section III-D-4
III-E-3-a Breeding of BSL-1 transgenic rodents Exempt under Section III-F-6, Appendix C-VII

Section III-F
Experiments Exempt from IBC Review but Require Registration

Section Subsection Research Application Description Comments
III-F Exempt experiments involve rDNA molecules that:
III-F-1 Are not in organisms or viruses
III-F-2 Consist entirely of DNA from single nonchromosomal or viral DNA source (one or more segments may be synthetic equivalent)
III-F-3 Consist entirely of DNA from prokaryotic host when propagated only in that host (or closely related strain of the same species) or when transferred to another host by well-established physiological means
III-F-4 Consist entirely of DNA from a eukaryotic host (excluding DNA from viruses) when propagated only in that host (or closely related strain of the same species)
III-F-5 Consist entirely of DNA from different species that exchange DNA by known physiological processes (one or more segments may be synthetic equivalent) See Appendix A for list of natural exchangers
III-F-6 Do not present significant risk to health or environment Appendix C
App C-1 rDNA containing < ½ of any eukaryotic viral genome in tissue culture Exceptions: Experiments described in Sections III-A or III-B, those involving RG3, 4, or restricted agents, large-scale experiments, or cloning of toxin molecule genes coding for biosynthesis of molecules toxic for vertebrates
App C-II E. coli K-12 host-vector systems Same exceptions as C-1
App C-III Saccharomyces host-vector systems Same exceptions as C-1
App C-IV Bacillus subtilis or Bacillus licheniformis host-vector systems Same exceptions as C-1
App C-V Extrachromosomal elements of listed gram positive organisms propagated and maintained in gram positive organisms See App C-V for listSame exceptions as C-1
App C-VI Purchase or transfer of transgenic rodents Only applies to rodents requiring BSL-1 containment
App C-VII Breeding of two different transgenic rodents or breeding of a transgenic rodent with a non-transgenic rodent Requirements:

1) Both parental rodents require BSL-1 containment,

2) Neither parental rodent contains incorporation of > ½ of the genome of an exogenous eukaryotic virus or incorporation of a transgene under control of a gammaretroviral long terminal repeat (LTR) and,

3) Rodent resulting from the breeding not expected to contain > ½ of an exogenous viral genome