Registration of Experiments Involving

Registration and Approval of rDNA Experiments

RECOMBINANT and SYNTHETIC NUCLEIC ACIDS

YALE BIOLOGICAL SAFETY COMMITTEE April 2019 (rev.)

This outline provides an overview of the “Guidelines for Research Involving Recombinant or Synthetic

Nucleic Acid Molecules” (NIH Guidelines). It is the responsibility of each investigator to make sure that

their laboratory is in compliance with these Guidelines. If your experiments require registration, check the

NIH Guidelines for the relevant regulatory section and the appropriate biosafety level or contact the

Biosafety Office or your Safety Advisor for assistance. For copies of the NIH Guidelines or rDNA

registration forms, please call Environmental Health & Safety (EHS) at 785-3550.

OEHS contacts: Phone: (203) 785-3550 Fax: 785-7588 Website: https://ehs.yale.edu/

Yale rDNA Forms and Information Regarding rDNA: https://ehs.yale.edu/recombinant-dna

NIH Office of Science Policy website: https://osp.od.nih.gov/biosafety-biosecurity-and-

emerging-biotechnology/

Experiments which must be

registered and approved prior to

initiation:

1. Deliberate transfer of a drug

resistance trait to a microorganism

(if it could compromise the use of

the drug to control disease agents in

human, animals, or agriculture);

2. Human gene transfer experiments;

3. Cloning DNA or RNA encoding

molecules lethal to vertebrates at an

LD50 of < 100 ug/kg body weight;

4. Experiments using human or animal

pathogens as host-vector systems;

5. Cloning of DNA or RNA from all

Risk Group 3, 4, or restricted

pathogens (includes HIV and human

tumor viruses), as well as Risk

Group 2 experiments involving ≥ 50

% of genetic material;

6. Recombinant DNA experiments

involving whole animals or plants:

7. Large-scale DNA work (i.e. > 10

liters of culture combined).

Examples:

1. Transferring a drug resistance trait that is used, had previously been used,

may be used (outside the U.S.), or that is related to other drugs that are

used to treat or control disease agents. Examples include: Transfer of

Erythromycin resistance into Borrelia burgdorferi; Transfer of

Pyrimethamine resistance into Toxoplasma gondii; Transfer of

Chloramphenicol resistance into Rickettsia conorii; Transfer of

Tetracycline resistance into Porphyromonas gingivalis.

2. Use of a defective adenoviral vector to deliver the CFTR gene

intranasally to patients with Cystic Fibrosis; Introduction of a HSV-TK

transduced cell line into patients with epithelial ovarian carcinoma,

followed by therapy with Gancyclovir.

3. Cloning toxins (or using plasmids that express toxins with low LD50’s)

such as Botulinum, Tetrodotoxin, Ricin, T-2, Saxitoxin, Abrin, Tetanus,

Shigella Dysenteriae, Pertussis, Staph Aureus Beta, Shiga Toxin, and

Conotoxins;

4. Use of pathogens or defective pathogen vectors (with or without helper

virus), such as Adenovirus, Adeno-Associated virus, Baculovirus, Herpes

virus, Lentivirus, Retrovirus, Vaccinia and Vesicular Stomatitis Virus.

5. rDNA experiments involving any quantity of genetic material from a Risk

Group 3 or higher pathogens (e.g., HIV, HTLV-1 & II, Prions,

Mycobacterium tuberculosis, West Nile Virus, Lymphocytic

Choriomeningitis Virus, and Rickettsia typhi. Note that rDNA

experiments involving ≥ 50 % of genetic material from Risk Group 2

organisms must also be registered with the IBC.

6. Creation of transgenic animals or plants (mice, rats, zebra fish,

drosophila, C. elegans etc.), or knockout animals that leave genetic

material in the animal as part of the silencing of the gene. Note: the

purchase (or transfer to your lab) of previously created transgenic rodents

is exempt from the regulations.

7. Use of a 10 L fermenter or growing up five 2 L flasks of rDNA culture

(i.e. E. coli K-12) qualifies as a large-scale experiment at Yale University.

NIH Guidelines Definitions and Information on

Recombinant or Synthetic Nucleic Acids

Section I-B. Definition of Recombinant and Synthetic Nucleic Acid Molecules

In the context of the NIH Guidelines, recombinant and synthetic nucleic acids are defined as:

(i) molecules that a) are constructed by joining nucleic acid molecules and b) that can replicate in a living

cell, i.e., recombinant nucleic acids;

(ii) nucleic acid molecules that are chemically or by other means synthesized or amplified, including those

that are chemically or otherwise modified but can base pair with naturally occurring nucleic acid

molecules, i.e., synthetic nucleic acids, or

(iii) molecules that result from the replication of those described in (i) or (ii) above.

Section III-C-1. Human gene transfer is the deliberate transfer into human research participants of either:

1.Recombinant nucleic acid molecules, or DNA or RNA derived from recombinant nucleic acidmolecules,

2.Synthetic nucleic acid molecules, or DNA or RNA derived from synthetic nucleic acidmolecules, that

meet any one of the following criteria:a.Contain more than 100 nucleotides; or

b.Possess biological properties that enable integration into the genome (e.g., ciselements involved in

integration); or

c.Have the potential to replicate in a cell; or

d.Can be translated or transcribed.

Synthetic Nucleic Acid Experiments that are covered by the Guidelines:

 Research that presents biosafety risks equivalent to rDNA research that is subject to the NIH

Guidelines such as research with a genetically modified virus or a vector derived solely by synthetic

techniques. Research involving synthetic nucleic acid molecules will require registration if:

 The molecules can replicate

 They can generate nucleic acids that can replicate in a living cell

 They can integrate into a host cell’s DNA

 They produce a toxin that is lethal for vertebrates at an LD50 of less than 100

nanograms/kilogram body weight

 They synthesize an organism that doesn’t occur naturally outside of a laboratory

setting (i.e. 1918 H1N1 Influenza)

 Human gene transfer experiments or clinical protocols with synthetic nucleic acid molecules if any

of the following criteria are met - the synthetic nucleic acid molecules:

 Contains more than 100 nucleotides; or

 Possess biological properties that enable integration into the genome (e.g. cis

elements involved in integration); or

 Have the potential to replicate in a cell; or

 Can be translated or transcribed.

Synthetic Nucleic Acid Experiments that are EXEMPT from the Guidelines:

 Introduction of certain synthetic nucleic acids into a biological system that is not expected to

present a biosafety risk that requires review by the IBC

 Introduction of synthetic nucleic acid molecules into biological systems akin to processes of nucleic

acid transfer that already occur in nature.

 Experiments with synthetic nucleic acid molecules that are not contained in cells, organisms or

viruses

 Those synthetic nucleic acid molecules that meet the following criteria shall be exempt:

1) Those that can neither replicate nor generate nucleic acids that can replicate in

any living cell (e.g. oligonucleotides or other synthetic that do not contain an

origin of replication or contain elements known to interact with either DNA or

RNA polymerase), and

2) Those that are not designed to integrate into DNA, and

3) Those that do not produce a toxin that is lethal for vertebrates at and LD50 of less

than 100 nanograms per kilogram body weight.