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Recombinant DNA Training University of Colorado at Colorado Springs

UNIVERSITY OF COLORADO AT COLORADO SPRINGS. Recombinant DNA Training University of Colorado at Colorado Springs. Department of Environmental Health and Safety www.uccs.edu/~pusafety/environmental/index.shtml. Recombinant DNA Training. Introduction.

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Recombinant DNA Training University of Colorado at Colorado Springs

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  1. UNIVERSITY OF COLORADO AT COLORADO SPRINGS Recombinant DNA Training University of Colorado at Colorado Springs Department of Environmental Health and Safety www.uccs.edu/~pusafety/environmental/index.shtml

  2. Recombinant DNA Training Introduction The purpose of this training module is to familiarize the Principal Investigator (PI) and lab personnel with the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines). These Guidelines are the standards for containment and safe research practices involving rDNA activities. After completing this training module, please complete the Recombinant DNA Quiz. Grades will be kept and tracked by Environmental Health and Safety.

  3. Recombinant DNA Training Introduction Links have been provided to direct you to specific Sections of these guidelines or to reference materials that can provide a more detailed description or explanation of the topic discussed. Principal Investigators are responsible for knowing the NIH Guidelines and training lab personnel as it pertains to your research.

  4. SCOPE OF THE NIH GUIDELINES SECTION I Purpose Applicability Compliance

  5. Recombinant DNA Training Purpose of NIH Guidelines The purpose of the NIH Guidelines is to specify practices for constructing and handling: • recombinant deoxyribonucleic acid (DNA) molecules, and • organisms and viruses containing recombinant DNA molecules.

  6. Recombinant DNA Training Definition of Recombinant DNA Molecules • In the context of the NIH Guidelines, recombinant DNA molecules are defined as either: (i) molecules that are constructed outside living cells by joining natural or synthetic DNA segments to DNA molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above. • Synthetic DNA segments which are likely to yield a potentially harmful polynucleotide or polypeptide (e.g., a toxin or a pharmacologically active agent) are considered as equivalent to their natural DNA counterpart. If the synthetic DNA segment is not expressed in vivo as a biologically active polynucleotide or polypeptide product, it is exempt from the NIH Guidelines. • Genomic DNA of plants and bacteria that have acquired a transposable element, even if the latter was donated from a recombinant vector no longer present, are not subject to the NIH Guidelines unless the transposon itself contains recombinant DNA.

  7. Recombinant DNA Training What are the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines) ? A document created in 1976 outlines principles for the safe conduct of research employing recombinant DNA (rDNA) technology. The NIH Guidelines detail practices and procedures for the containment of various forms of recombinant DNA research, for the safe conduct of research involving genetically modified plants and animals, and for the safe conduct of human gene transfer research. As a living document, it is periodically revised to keep pace with the changing state of science. http://oba.od.nih.gov/rdna/nih_guidelines_oba.html

  8. Recombinant DNA Training What is the NIH Office of Biotechnology Activities (NIH-OBA)? What is the NIH Office of Biotechnology Activities (NIH-OBA)? The NIH Office of Biotechnology Activities (NIH-OBA) promotes science, safety, and ethics in biotechnology through the advancement of knowledge, enhancement of public understanding, and development of sound public policies. A core responsibility of NIH-OBA is to foster awareness of, and adherence to, the standards and practices set forth in the NIH Guidelines.

  9. Recombinant DNA Training NIH Recombinant DNA Advisory Committee (RAC) • Federal advisory committee providing advice and recommendations to the NIH Director regarding rDNA research • Unique public forum for the discussion of science, safety, and ethics of rDNA research • Reviews and analyzes unique clinical gene transfer protocols and safety information • Observations and findings of general importance to the field • Not an approved process

  10. Recombinant DNA Training What is an Institutional Biosafety Committee (IBC)? Institutional Biosafety Committees (IBCs) provide local review and oversight of nearly all forms of research utilizing recombinant DNA. They ensure that recombinant DNA research conducted at or sponsored by the institution is in compliance with the NIH Guidelines. A requirement of the NIH Guidelines is that an IBC must review and approve all research subject to the NIH Guidelines.

  11. Recombinant DNA Training University of Colorado Colorado Springs IBC The Institutional Biosafety Committee (IBC), exercises oversight for all University of Colorado at Colorado Springs (UCCS) activities involving biological agents or materials, to ensure that employees, students, the public and the environment are protected from biohazards associated with UCCS operations. This includes all educational and research laboratories, and field research. The IBC also reviews and approves research that is conducted by UCCS faculty and students at other institutions and non-CU entities. UCCS’s IBC meets annually. The IBC application, forms, meeting schedule and application submission deadlines are posted on the UCCS Sponsored Programs website. http://www.uccs.edu/~osp/.

  12. Recombinant DNA Training Who Must Comply with the NIH Guidelines All institutions that receive NIH funding for recombinant DNA research must comply with the NIH Guidelines. Researchers at institutions that are subject to the NIH Guidelines must comply with the requirements even if their individual projects are not funded by NIH.

  13. Recombinant DNA Training What are Potential Consequences of Noncompliance with the NIH Guidelines for NIH Funded Projects? • For NIH funded projects involving rDNA techniques: • suspension, limitation, or termination of NIH funds for the noncompliant NIH-funded research project and of NIH funds for other rDNA research at the institution, or that of the Principal Investigator, or • a requirement for prior NIH approval of any or all rDNA projects at the institution.

  14. Recombinant DNA Training What are Potential Consequences of Noncompliance with the NIH Guidelines for Non-NIH Funded Projects? • For Non-NIH funded projects involving rDNA techniques conducted at or sponsored by an institution that receives NIH funds for projects involving such techniques: • suspension, limitation, or termination of NIH funds for rDNA research at the institution, or that of the Principal Investigator, or • a requirement for prior NIH approval of any or all rDNA projects at the institution.

  15. SAFETY CONSIDERATIONS SECTION II Risk Assessment Risk Groups Containment

  16. Recombinant DNA Training Risk Assessment and Risk Groups (RG) The Guidelines require that the Principal Investigator (PI) make an initial assessment of risk based on the Risk Group (RG) of an agent (see Appendix B of the Guidelines, Classification of Human Etiologic Agents on the Basis of Hazard). This is based on the World Health Organization (WHO) Risk Classification.

  17. Recombinant DNA Training Basis for the Classification of Biohazardous Agents by Risk Group (RG)

  18. Recombinant DNA Training Containment Requirements • The Biosafety Officer (BSO) and the IBC will review the agent itself and the proposed experiment procedures to determine the appropriate level of containment. • The levels of containment or Biosafety Levels are described in Appendix G of the Guidelines http://oba.od.nih.gov/oba/rac/guidelines_02/APPENDIX_G.htm and are also found in the Centers for Disease Control and Prevention (CDC) guideline, Biosafety in Microbiological and Biomedical Laboratories, 5th ed http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm

  19. EXPERIMENTS COVERED BY THE NIH GUIDELINES SECTION III Experiments that require IBC Approval Section III-A through Section III-F

  20. Recombinant DNA Training Experiments Covered by the NIH Guidelines: Section III-A Experiments that Require IBC Approval, RAC Review, and NIH Director Approval Before Initiation Section III-B Experiments That Require NIH/OBA and IBC Approval Before Initiation Section III-C Experiments that Require IBC and Institutional Review Board Approvals and RAC Review Before Research Participant Enrollment Section III-D Experiments that Require IBC Approval Before Initiation Section III-E Experiments that Require IBC Approval Simultaneous with Initiation Section III-F Exempt Experiments

  21. Recombinant DNA Training Recombinant DNA ExperimentsSection III-A of the NIH Guidelineshttp://oba.od.nih.gov/oba/rac/guidelines_02/NIH_Guidelines_Apr_02.htm#_Toc7261560 • This category is limited to studies that involve the deliberate transfer of drug resistance to microorganisms (not known to acquire the trait naturally) that can compromise the use of the drug to control the microorganism and its disease in humans, veterinary medicine or agriculture, e.g., a recombinant HBV virus that was would render the current HBV vaccine series as ineffective. • These experiments require IBC approval, RAC Review, and NIH Director approval before initiation and is considered a Major Action.

  22. Recombinant DNA Training Recombinant DNA ExperimentsSection III-B of the NIH Guidelineshttp://oba.od.nih.gov/oba/rac/guidelines_02/NIH_Guidelines_Apr_02.htm#_Toc7261562 • This category is limited to cloning of genes that encode for toxin molecules with LD50 <100 nanograms/kg body weight (e.g., botulinum, tetanus, diphtheria toxins). • These experiments involve the potential manipulation of sequences from microbial toxins such as the botulinum toxins, tetanus toxin, diphtheria toxin, and Shigella dysenteriae neurotoxin, e.g., creation of recombinant organism, such as E. coli K12 with a sequence to synthesize Clostridium botulinum toxin. • These experiments require NIH-OBA and IBC approval before initiation (may require CDC Select Agent registration if exempt quantities are not used).

  23. Recombinant DNA Training Recombinant DNA ExperimentsSection III-Cof the NIH Guidelineshttp://oba.od.nih.gov/oba/rac/guidelines_02/NIH_Guidelines_Apr_02.htm#_Toc7261564 • This category is limited to deliberate transfer of recombinant DNA, or DNA or RNA derived from recombinant DNA, into one or more human subjects (e.g. in-vivo human gene therapy). • These experiments often involve in-vivo human gene therapy, but may also include vaccine studies, e.g. an adenoviral mediated gene transfer experiment. • These experiments require IBC and IRB approvals and RAC Review before research participant enrollment.

  24. Recombinant DNA Training Recombinant DNA ExperimentsSection III–D of the NIH Guidelineshttp://oba.od.nih.gov/oba/rac/guidelines_02/NIH_Guidelines_Apr_02.htm#_Toc7261566 There are 7 subcategories for Section III-D: Section III-D-1 Experiments Using Risk Group 2, Risk Group 3, Risk Group 4, or Restricted Agents as Host-Vector Systems (see Section II-A, Risk Assessment) Section III-D-2 Experiments in which DNA from Risk Group 2, Risk Group 3, Risk Group 4 or restricted agents is cloned into nonpathogenic prokaryotic or lower eukaryotic host-vector systems • Experiments in this category require IBC approval before initiation

  25. Recombinant DNA Training Recombinant DNA ExperimentsSection III–D of the NIH Guidelineshttp://oba.od.nih.gov/oba/rac/guidelines_02/NIH_Guidelines_Apr_02.htm#_Toc7261566 There are 7 subcategories for Section III-D, continued: Section 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 Section III-D-4 Experiments involving whole animals – the animal’s genome has been altered by the stable introduction of rDNA, or RNA derived therefrom, into the germ line (transgenic animals), and Experiments involving viable rDNA-modified microorganisms tested on whole animals

  26. Recombinant DNA Training Recombinant DNA ExperimentsSection III–D of the NIH Guidelineshttp://oba.od.nih.gov/oba/rac/guidelines_02/NIH_Guidelines_Apr_02.htm#_Toc7261566 There are 7 subcategories for Section III-D, continued: Section III-D-5 Experiments to genetically engineer plants by recombinant DNA methods, to use such plants for other experimental purposes (e.g., response to stress), to propagate such plants, or to use plants together with microorganisms or insects containing rDNA. Section III-D-6 Experiments involving more than 10 liters of culture. The appropriate containment will be determined by the IBC. Section III-D-7 Experiments involving influenza viruses.

  27. Recombinant DNA Training Recombinant DNA ExperimentsSection III–E of the NIH Guidelineshttp://oba.od.nih.gov/oba/rac/guidelines_02/NIH_Guidelines_Apr_02.htm#_Toc7261573 • This category includes experiments that are not included in Sections III-A through III-D, III-F and their subcategories are considered in Section III-E. These experiments may be conducted at BL-1 containment. • These experiments require IBC approval simultaneous with initiation. • There are 3 subcategories for Section III-E: • Section III-E-1 Experiments involving the formation of rDNA molecules containing no more than two-thirds of the genome of any eukaryotic virus.

  28. Recombinant DNA Training Recombinant DNA ExperimentsSection III–E of the NIH Guidelineshttp://oba.od.nih.gov/oba/rac/guidelines_02/NIH_Guidelines_Apr_02.htm#_Toc7261573 There are 3 subcategories for Section III-E: Section III-E-1 Experiments involving the formation of rDNA molecules containing no more than two-thirds of the genome of any eukaryotic virus. Section III-E-2 This section covers experiments involving rDNA- modified whole plants, and/or experiments involving recombinant DNA-modified organisms associated with whole plants, except those experiments that fall under Section III-A through III-D. BL-1 containment is adequate.

  29. Recombinant DNA Training Recombinant DNA ExperimentsSection III–E of the NIH Guidelineshttp://oba.od.nih.gov/oba/rac/guidelines_02/NIH_Guidelines_Apr_02.htm#_Toc7261573 There are 3 subcategories for Section III-E, continued: Section III-E-3 Experiments involving transgenic rodents. This section covers experiments involving the generation of rodents in which the animal's genome has been altered by stable introduction of recombinant DNA, or DNA derived therefrom, into the germ-line (transgenic rodents). Only experiments that require BL1 containment are covered under this section; experiments that require BL2, BL3, or BL4 containment are covered under Section III-D-4, Experiments Involving Whole Animals.

  30. Recombinant DNA Training Recombinant DNA ExperimentsSection III–F of the NIH Guidelineshttp://oba.od.nih.gov/oba/rac/guidelines_02/NIH_Guidelines_Apr_02.htm#_Toc7261577 • Experiments involving rDNA molecules included in this category are exempt from these Guidelines. However, the IBC at University of Colorado requires that an IBC application be submitted for review and approval. • There are 6 subcategories for Section III-F. • These experiments require IBC approval simultaneous with initiation.

  31. Recombinant DNA Training Other Institutional Committees and the IBC Remember that if you submit a protocol to the IACUC or IRB and it involves rDNA techniques or other biological materials, you must also submit an IBC application for review and approval of this work. IRB IACUC IBC

  32. ROLES AND RESPONSIBILITIES SECTION IV IBC Biosafety Officer Principal Investigator

  33. Recombinant DNA Training IBC Responsibilities RequirementReference • Adequate IBC Training Section IV-B-1-h • Annual Report To NIH Section IV-B-2-a-(3) • No Conflict of Interest Section IV-B-2-a-(4) • Write Meeting Minutes Section IV-B-2-a-(7)

  34. Recombinant DNA Training IBC Responsibilities AssignmentReference • Assess Lab Facilities Section IV-B-2-b-(1) • Notify PI of Approval Section IV-B-2-b-(2) • Set Containment Level Section IV-B-2-b-(3-4) • Monitoring Projects Section IV-B-2-b-(5) • Establish Emergency Plans Section IV-B-2-b-(6) • Notify NIH-OBA & UCB of Section IV-B-2-b-(7) Accidents, Illnesses, & NIH Violations within 30 days • Ensure NIH review Section IV-B-2-b-(8)

  35. Recombinant DNA Training Biosafety Officer (BSO) Responsibilities AssignmentReference • Conduct Inspections Section IV-B-3-c-(1) • Notify IBC & UCCS of Section IV-B-3-c-(2) Accidents, Illnesses, & NIH Violations • Investigate Accidents Section IV-B-3-c-(3) • Establish Emergency Plans Section IV-B-3-c-(3) • Advise on Lab Security Section IV-B-3-c-(4) • Provide Technical Advice Section IV-B-3-c-(5)

  36. Recombinant DNA Training Principal investigator Responsibilities Principal Investigators (PIs) are responsible for full compliance with the NIH Guidelines during the conduct of recombinant DNA research. As part of this general responsibility, the PI should: • Be adequately trained in good microbiological techniques. • Provide laboratory research staff with protocols describing potential biohazards and necessary precautions. • Instruct and train laboratory staff in: (i) the practices and techniques required to ensure safety, and (ii) the procedures for dealing with accidents. • Inform the laboratory staff of the reasons and provisions for any precautionary medical practices advised or requested (e.g., vaccinations or serum collection).

  37. Recombinant DNA Training Principal investigator Responsibilities • Supervise laboratory staff to ensure that the required safety practices and techniques are employed. • Correct work errors and conditions that may result in the release of recombinant DNA materials. • Ensure the integrity of physical containment (e.g., biological safety cabinets) and biological containment (e.g., purity and genotypic and phenotypic characteristics). • Comply with permit and shipping requirements for recombinant DNA molecules. • Adhere to IBC-approved emergency plans for handling accidental spills and personnel contamination.

  38. Recombinant DNA Training Principal investigator Responsibilities Before initiating research subject to the NIH Guidelines, the PI must: • Determine whether the research is subject to Section III-A, III-B, III-C, III-D, III-E, or III-F of the NIH Guidelines. • Propose physical and biological containment levels in accordance with the NIH Guidelines when registering research with the IBC. • Propose appropriate microbiological practices and laboratory techniques to be used for the research. • Submit an IBC application to the IBC for review and approval.

  39. Recombinant DNA Training Principal investigator Responsibilities • Seek OBA’s determination of containment for experiments that require case-by-case review. • Petition OBA, with notice to the IBC, for proposed exemptions from the NIH Guidelines. • Obtain IBC approval before initiating research subject to the NIH Guidelines. • Seek NIH approval, in addition to IBC approval, to conduct experiments specified in Sections III-A and III-B of the NIH Guidelines.

  40. Recombinant DNA Training Principal investigator Responsibilities While conducting research subject to the NIH Guidelines, the PI must: • Determine the need for IBC review before modifying rDNA research already approved by the IBC. • Submit any subsequent changes (e.g., changes in the source of DNA or host-vector system) to the IBC for review and approval or disapproval. • Remain in communication with the IBC throughout the duration of the project. • Report any significant problems pertaining to the operation and implementation of containment practices and procedures, violations of the NIH Guidelines, or any significant research-related accidents (e.g. needlestick) and illnesses to the Biological Safety Officer, the IBC, NIH-OBA, Greenhouse or Animal Facility Director (where applicable), and other appropriate authorities within 30 days.

  41. THE KEY APPEDICES Appendix B Appendix G Appendix K Appendix P Appendix Q

  42. Recombinant DNA Training Appendix BCLASSIFICATION OF HUMAN ETIOLOGIC AGENTS ON THE BASIS OF HAZARD This appendix includes those biological agents known to infect humans as well as selected animal agents that may pose theoretical risks if inoculated into humans. Included are lists of representative genera and species known to be pathogenic; mutated, recombined, and non-pathogenic species and strains are not considered. Non-infectious life cycle stages of parasites are excluded. The WHO Risk Group classification is used in the guidelines.

  43. Recombinant DNA Training Appendix GPHYSICAL CONTAINMENT Appendix G specifies physical containment for standard laboratory experiments and defines Biosafety Level 1 - Biosafety Level 4, consistent with the CDC Guideline, Biosafety in Microbiological and Biomedical Laboratories. The following appendices shall be used to determine containment in the following instances: • For large-scale (over 10 liters) research or production, Appendix K shall supersede Appendix G. • For certain work with plants, Appendix P shall supersede Appendix G. • For certain work with animals, Appendix Q supersedes Appendix G.

  44. Recombinant DNA Training Appendix KLARGE SCALE CONSIDERATIONS • Appendix K specifies physical containment guidelines for large-scale (greater than 10 liters of culture produced at one time) research or production involving viable organisms containing recombinant DNA molecules. • It shall apply to large-scale research or production activities as specified in Section III-D-6, Experiments Involving More than 10 Liters of Culture. • For the purpose of large scale research or production, four levels of containment are established. These are referred to as BL1-Large Scale (LS), BL2-LS, BL3-LS, and Good Large Scale Practice (GLSP)

  45. Recombinant DNA Training Appendix PPHYSICAL AND BIOLOGICAL CONTAINMENT FOR rDNA RESEARCH INVOLVING PLANTS • Appendix P specifies physical and biological containment conditions and practices suitable to the greenhouse conduct of experiments involving recombinant DNA-containing plants, plant-associated microorganisms, and small animals and will supersede Appendix G when the research plants are of a size, number, or have growth requirements that preclude the use of containment conditions described in Appendix G. • Four biosafety levels (referred to as Biosafety Level 1 – Plants or BL1-P, BL2-P, BL3-P, and BL4-P) are used for research involving recombinant DNA molecules in plants requiring special containment.

  46. Recombinant DNA Training Appendix QPHYSICAL AND BIOLOGICAL CONTAINMENT FOR rDNA RESEARCH INVOLVING ANIMALS • Appendix Q specifies containment and confinement practices for research involving whole animals, both those in which the animal's genome has been altered by stable introduction of recombinant DNA, or DNA derived therefrom, into the germ-line (transgenic animals) and experiments involving viable recombinant DNA-modified microorganisms tested on whole animals. • For the purpose of animal research, four levels of containment are established in Appendix Q. These are referred to as BL1-Animals (N), BL2-N, BL3-N, and BL4-N.

  47. WHAT DOES ALL OF THIS MEAN TO ME?

  48. Recombinant DNA Training Apply To The IBC • If you are conducting any work with rDNA molecules, then you must complete an IBC application, regardless of your source of funding and even if it meets the “Exempt” classification of the Guidelines. • The University reviews alI lab, field, and class activities involving biological agents and materials. • Currently, UCCS does not allow possession or use of Risk Group 3 or 4 agents which would require BL-3 or BL-4 containment. • Currently, UCCS does not allow possession or use of Select Agents or Toxins that would require registration with the CDC or USDA.

  49. Recombinant DNA Training IBC Application Research Description • The “Research Description” is one of the most important parts of the IBC Application because it helps the IBC assess the appropriate level of containment to safely work with the agents and materials listed. • It’s important that you include details about how each biological agent or material will be used, e.g. experiment procedure(s), agent strain(s), etc.

  50. Recombinant DNA Training IBC Application Which Section(s) of the Guidelines does my research fall under? • Based on the agent used, e.g. adenovirus 5, determine what risk group it is by referring to Appendix B of the Guidelines. In our example of adenovirus, adenovirus is a Risk Group 2 organism. • Once the Risk Group of the agent is known, determine the type of experiment described in Section III of the NIH Guidelines (e.g. Section III-D-1, the use of Risk Group 2 agents as part of a host vector system).

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