Biological Use Authorization (BUA) Application FAQs

Q1. What information should I provide in Question 2?

Provide a brief but comprehensive description of the actual experimental procedures and biohazardous agents including rDNA that will be used for the proposed research. You are welcome to use a narrative or other formats as you wish, but all research involving biohazards needs to be accounted for in this question. You are strongly encouraged to use general terms for common laboratory procedures such as the following:

• Engineering of plasmids in laboratory strain E. coli
• Culture of human cell lines
• Sorting human cells by flow cytometry

However, also be specific about which biohazards will be associated with which procedures. Some examples include the following.

• Breeding mice containing the transgene for GFP
• Transducing human cell lines with an adenoviral vector expressing the gene MYC
• Working with a lentiviral vector expressing a GFP reporter gene

Be sure to specifically mention procedures that preclude the use of physical containment that would otherwise be required (e.g., stereotactic injections if Risk Group 2 viral vectors to animals, imaging of animals exposed to human cells when the imaging equipment cannot fit inside a biosafety cabinet). You may want to complete this question after you have filled out the rest of the application to ensure that all relevant procedures and biohazardous materials/agents are included. Be aware that your application may be returned to you as incomplete if this question is not consistent with the rest of your application.

Q2. What information should I provide about my work involving rDNA in Question 2?

If your research includes rDNA from any portion of one or a few genes, explicitly list the genes as well as the context in which the DNA or RNA will be used. If your research involves a large number of genes, then describe the functional categories of the proteins encoded by these genes, such as 'cytokine receptors' or 'cell signaling', as well as the context in which they will be used. If your research includes the discovery of new genes that will then be used in your research, describe the functional properties of the proteins encoded by the target genes (if known), how they will be identified (using general terms), and what you will do with them once they are identified.

You MUST use RefSeq gene names when listing specific genes. This is essential for reviewers to carry out a risk assessment of your proposed research. Be aware that your application may be returned to you as incomplete if you do not use RefSeq gene names. Instructions for verifying correct RefSeq gene names are included in the FAQ for question 36B.

Q2. What information should I not include in Question 2?

Do not provide experimental protocols or details such as dosing regimens or specific concentrations. Do not cut and paste procedures from animal protocols, grant proposals, or standard operating procedures. Do not provide the scientific justification, rational, or hypotheses associated with the proposed research.

Q1. What information should I provide in Question 3?

Please describe what elements of your research that you believe, in your professional opinion, pose the greatest biohazardous risk to laboratory personnel or the environment. Be aware that a formal review of your application may lead to the identification of other biohazards associated with the proposed research which, in the opinion of biosafety professionals or members of the IBC, may constitute an even greater hazard. If this is the case, you may be contacted for further discussion on the topic. Be aware that your application may be returned to you as incomplete if you fail to identify what you believe to be the greatest biohazard associated with your proposed research.

Q23. I work only with established human cell lines obtained from a commercial vendor (e.g., ATCC) which is a very reliable and authentic source. I don't think the Washington State BBP rule applies to me. Please confirm.

The Washington State BBP Rule applies to all work done with human blood, tissue, body fluids visibly contaminated with blood or other potentially infectious materials (OPIM), as well as human cells and cell lines. All human cell lines, whether established or from authentic sources, are covered under the Washington State BBP Rule due to their potential to harbor BBPs. For a list of OPIM and information about the BBP program, refer to EH&S Bloodborne Pathogens. 

Q23. What information should I provide if I work with human tissue, blood, or body fluids, or culture of human primary cells or cell lines?

If you answered YES to questions 11, 15, 16, 19 and/or 20 for work with human blood, tissue, or body fluids, culture of human primary cells or cell lines, or iPSCs, the Washington State BBP Rule applies. As part of the BBP program, you will need to submit a site-specific BBP Exposure Control Plan. Initial and annual BBP training is also required. For more information, refer to Bloodborne Pathogens.

Q31-34. Select Agents

I work with an excluded or attenuated strain of a Select Agent. Can you provide some examples of the rationale you would like for why the strain is exempt?

Select Agent excluded and attenuated strain information can be found on the CDC Select Agents and Toxins Exclusions website.

Examples of the rationale for an excluded or an attenuated Select Agent include:

• A/Vietnam/1203 (H5N1) PR8 is excluded from the Select Agent regulations under Section 121.3 (e). This strain contains segments from A/PR/8/34 (H1N1) and H5 and N1 from Highly Pathogenic Avian Influenza A/Vietnam/1203/2004 (H5N1). The polybasic cleavage site in HA has been mutated to render it less pathogenic.  
• Venezuelan equine encephalitis (VEE) subtypes ID and IE are excluded from the Select Agent regulations under Section 73.4 (e) and 121.4 (e).
• Venezuelan equine encephalitis virus (VEEV) strain TC-83 excluded from the Select Agent regulations under Section 73.4 (e) and 121.4 (e). The strain is a vaccine strain of VEE.
• West African clade of Monkeypox virus is excluded from the Select Agent regulations under Section 73.3 (e). Clinically severe human disease associated with West African strains is rare, and this virus clade has not been associated with human mortality.
• Ebola ΔVP30 is excluded from the Select Agent regulations under Section 73.3 (e). This virus lacks the gene encoding for the VP30 protein. Therefore, the virus is replication incompetent and does not form infectious progeny in wild-type cells and does not cause disease in animals.

Q33. I work with nucleic acids from select agents. Do I need to complete this section?

Yes, complete this section if you work with or propose to work with positive strand RNA forms of Select Agent viral genomes or nucleic acids encoding for the toxic form(s) of regulated Select Toxins.

Q45. I work with infectious clones (e.g., cDNA clones), should I list them here?

Yes, work with cDNA clones needs to be listed in this table. Also describe your infectious clones and what you will be doing with them in Q38d.

Q45B. What are the different replication status options?

• Replication defective and testing for RCV: Most viral vectors are engineered so that the viral nucleic acid packaged into the viral vector particle and transferred to recipient cells is insufficient to support replication of the vector and production of infectious progeny. Note that the NIH Guidelines (Section III-D) defines defective viruses as those containing less than 2/3 of the genome of any eukaryotic virus where all viruses from the same family are considered identical. Production of replication defective vectors requires complementation of essential elements in trans, often in the form of plasmids or packaging cells. Therefore, production of replication defective vectors often involves an inherent risk of reconstituting an intact or replication-competent virus (RCV), because all the genetic elements needed to produce infectious progeny may be found together in the producer cells. Some vector systems (e.g., third generation or later lentiviral vectors) incorporate a sufficiently high level of engineering safeguards to render the production of RCV almost impossible. However, for other commonly used viral vectors like amphotropic gammaretroviruses, RCV could persist undetected in transduced cells or animals indefinitely. This is a particular concern for vectors encoding oncogenes. Thus, in addition to the characteristics of the vector and the identity of the transgene, RCV testing may inform the appropriate biosafety levels for the use of viral vectors as well as cells, animals, and plants exposed to viral vectors. RCV testing varies depending on the viral vector, but common techniques include PCR tests for reconstitution of the genome, plaque assays for viral replication and spread, or detection of viral gene products (e.g., by ELISA). If RCV testing is performed, the results must be included with the BUA to be incorporated into the risk assessment.
• Replication competent: Some applications require viral vectors capable of infecting cells and replicating. One example is the use of oncolytic vectors for cancer treatment. In this case, the genome may be modified so that viral replication occurs preferentially in certain cell types (e.g., transformed cells). Alternatively, or in addition, a transgene may be added to the genome in a way that does not interfere with replication. Information about the conditions under which the vector will replicate, any attenuation of pathogenicity or modification of tropism compared to the wild type virus, as well as the identity of the transgene are all important elements of risk assessment for these vectors.
• Negative for replication competent virus (RCV): Selecting this indicates that you have had your viral vector or transduced cell line appropriately testing for the presence of RCV and the testing showed RCV is not present. If RCV testing has been performed on viral vectors, submit the RCV test results with the BUA application. More information about which viral vectors can be RCV tested and the appropriate tests is on our Viral Vectors for Gene Transfer webpage.
• Other (describe): This is an option if needed for special circumstances.
• Not applicable (non-viral): This applies for non-viral gene delivery methods such as plasmids, mRNA, siRNA, etc.

Q45C. I am asked to provide gene inserts or transgenes. Can I use gene names other than RefSeq gene names in column C of the Viral Vector and Gene Delivery Methods table?

No, gene names other than RefSeq gene names may not be used in this column. The IBC requires that you provide only RefSeq gene names. Some examples of RefSeq gene names include 'GFP', 'MYC', and 'COP4'. To verify that your gene names are correct follow the instructions below.

1. Access the National Center for Biotechnology Information (NCBI) RefSeqGene website.
2. Enter your gene name. Select 'Search' to search the RefSeq database.
3. The search results for your gene will display. If your gene name appears as blue linked text, you have entered a correct RefSeq gene name.

If your gene is a synthetic construct and does not appear in RefSeq, provide a reference that describes the gene.

If your research involves more genes than can be easily listed in column C, list the functional categories or the categories of the encoded proteins in Question 48. 

Q45D. My project involves in vitro work using viral vectors. How should I complete column D of the Viral Vector and Gene Delivery Methods table?

If your project involves in vitro work, mark YES in column D. In your answer, specify the cell species/type in which the viral vector (or other rDNA) will be used and explain the activities. Refer to examples provided on the Viral Vectors and Gene Delivery Methods table.

Q45E. My project involves administering viral vectors to animals. How should I complete column E of the Viral Vector and Gene Delivery Methods table?

If your project involves in vivo work, mark YES in column D. In your answer, specify the animal species, method of administration, exactly what will be administered to the animal, and explain the activities. For example:

• Third generation lentiviral vectors will be injected into mouse footpad
• Human cells transduced with amphotropic gammaretroviral vectors will be xenografted onto rat forelimbs
• mRNA vaccines will be administered to mice via IP injection

Q47. What are third generation lentiviral vectors? Why do I need to provide additional information for these vectors?

The IBC has established a working definition of third generation lentiviral vectors. If vectors meet the definition of third generation, biocontainment may be lowered for use with oncogenic inserts. Refer to Third Generation Lentiviral Vectors for more information.

Third generation lentiviral vector systems improve on the safety of earlier lentiviral vector systems by splitting the necessary genes onto additional plasmids which renders them essentially incapable of generating replication-competent virus. The packaging system is split into two plasmids: one encoding Rev and one encoding Gag and Pol. Then Tat is eliminated from the third generation system through the addition of a chimeric 5' LTR fused to a heterologous promoter on the transfer plasmid. Thus, expression of the transgene from this promoter is no longer dependent on Tat transactivation. Finally, the third generation transfer plasmid can be packaged by either a second generation or third generation packaging system.

For more information on third generation lentiviral vectors and how they compare to second generation lentiviral vectors, refer to the Addgene Lentiviral Guide.

If you are unable to identify all four plasmids in your lentiviral vector system, the lentiviral vector will be listed as first/second/unknown generation on your BUA application.

Q49-52. How do I complete Questions 49-52?

You are required to disclose the use of all oncogenes and tumor suppressor genes in this section. Follow these instructions to complete this section.

To search the two databases:

1. Verify that you are using correct RefSeq gene names. Instructions for verifying correct RefSeq gene names are included in FAQ for Q45C.
2. Using RefSeq gene names, search both databases for the gene and review the information:
   
   1. For the Cancer Gene Census database, search using the search box in the Census table, not the search box on the site's toolbar.
   2. For the Cancer Genetics Web database, you can find genes using the alphabetical list or use the search field.
3. If any of your genes appear in the Cancer Gene Census database or are described as oncogenes or tumor suppressors in the Cancer Genetics Web database, mark YES to Question 49 and list the gene names.
4. If your genes do not appear in the database but are well described in the scientific literature as oncogenes or tumor suppressors, mark YES to Question 50. In your answer, list the gene names, briefly describe why they are considered oncogenes, and provide references.
5. If your genes do not appear in the database but you have reasons to believe they are oncogenes or tumor suppressors, mark YES to Question 51. In your answer, list the gene names, briefly describe why you consider them to be oncogenes or tumor suppressors, and provide references.

Q52. What is the point of Question 52? I believe my work with oncogenes or tumor-suppressor genes should not require an elevation in biocontainment, and I do not want to work at an elevated level of biocontainment. Can I petition the IBC to not increase the level of biocontainment for my work with these oncogenes or tumor-suppressor genes? 

If any of the genes you are using are identified as potential oncogenes or tumor suppressors in the previous questions, describe in Q52 whether you are overexpressing or dysregulating oncogenes or knocking down or silencing tumor suppressors. The IBC will consider this when setting the biosafety level for this work.

It is the IBC's policy that the addition of oncogenes or tumor suppressor genes to certain classes of viral vectors necessitates that work with these vectors is carried out at higher levels of biocontainment. This includes work with oncogenes or tumor suppressor genes in all classes of vectors based on retroviruses, adeno-associated viruses, and any other virus-based vectors that have a reasonable capacity for genomic integration.

Give a description of the type of expression for each oncogene or tumor suppressor in each vector system. This will help to determine the biocontainment level.

Examples include:

1. I am overexpressing oncogene X.
2. I am overexpressing tumor suppressor gene X.
3. I am knocking down the expression of oncogene X.
4. I am silencing tumor suppressor gene X.
5. I am working with an oncogene but have deleted a functional domain known to be necessary for the oncogenic potential of the corresponding protein.
6. I am working with an oncogene that appears in one of the oncogene databases, but there is scientific evidence that this gene is not a driver of oncogenesis.

Please provide references from scientific literature as applicable.

Q54. I am asked to identify the containment requirements and biosafety levels of my laboratory. Can you explain the different biocontainment levels?

Each Principal Investigator (PI) is responsible for performing a risk assessment of their work to identify hazards. The risk assessment helps determine which biohazard containment and laboratory practices are appropriate. Refer to Biological Risk Assessment for more information, including Risk Groups and Biological Safety Levels and the associated references.

Biosafety levels are well described by in the CDC’s BMBL and in the NIH Guidelines:

1. Laboratory biosafety levels (BSLs) refer to in vitro lab spaces or shared facilities where biohazardous agents are used. The four BSLs consist of combinations of facility design features, primary and secondar barriers, practices and procedures, and personal protective equipment (PPE).
2. Animal facility biosafety levels (ABSLs) refer to labs or animal facility spaces where biohazardous agents and rDNA are used in animals or where transgenic animals are used. ABSLs correspond to increased levels of protection for research staff and the environment and are recommended as the minimal standard for activities involving laboratory animals exposed to biohazardous agents. The animal care and use environment can present unique hazards not found in standard research laboratories including bites and scratches, zoonotic disease, shedding of infectious agents, etc. Keeping this in mind, all additional animal facility standard operating procedures must be followed.
3. Plant facility biosafety levels (BSL-Ps) refer to labs or greenhouse spaces where biohazardous agents are used with plants or where transgenic plants are used. There are specialized requirements for research involving transgenic plants or biohazardous agents used with plants. More information is available on the Plant Research Biosafety webpage.
4. Arthropod biosafety levels (ACLs) refer to labs or facilities where arthropods are used. Arthropod facilities may be classified according to biohazardous agents used in arthropods or based on the risk or hazard of the arthropod itself. Contact EH&S Biological Safety if you are unsure what ACL applies to your lab.

If this question is completed incorrectly, a biosafety officer will notify you and help you to determine the correct biosafety levels for your laboratory.

Q55-57. I am asked to list biohazardous agents and activities. Since I already listed the activities in previous questions, do I need to list them here again? If so, what do I need to list?

Yes, please provide a summary of the biohazardous agents and the general activities that will be done in each room. Examples of what should be listed include:

• Culture of human cell lines, E. coli, 3rd gen. lentiviral vectors, and Pseudomonas aeruginosa. Sequencing and analysis of clinical P. aeruginosa isolates.
• Human and NHP cell infections with RG2 viruses. Culture of RG2 viruses and plasmid generation in E. coli K12 strains.
• Aerosol exposure of mice to bacteria and administration of a recombinant DNA vaccine to mice.
• Intranasal inoculation of mice to RG2 bacteria.
• Cell sorting of human and non-human primate cells, imaging of mice exposed to lentiviral vectors, generation of human iPS cells.

Q56. I am asked to identify the biosafety levels of my animal facilities. I am not sure about the biocontainment levels for my animal work. Can you explain the different animal biocontainment levels?

Animal Biosafety Level (ABSL) classification refers to facilities, practices, and operational requirements applicable to work with animals that have been exposed to biohazardous agents. Animals exposed to biohazardous agents assigned to BSLs 1-4 are generally assigned into ABSLs 1-4, respectively. ABSLs correspond to increased levels of protection for research staff and the environment and are recommended as the minimal standard for activities involving laboratory animals exposed to biohazardous agents. Note that no ABSL-4 research is conducted at the UW. Refer to the Animal Activities Table from NIH Office of Science Policy for a listing of animal experiments covered under the NIH Guidelines with references to the corresponding NIH Guidelines sections and biosafety levels.

Practices and facility requirements for biosafety laboratories apply to research with animals as well. The animal care and use environment can present unique hazards not found in standard microbiological research laboratories (e.g., animals may generate aerosols, bite and scratch, be infected with a zoonotic agent, etc.) Keeping this in mind, all additional animal facility standard operating procedures must be followed.

If this question is completed incorrectly, a biosafety officer will notify you and help you to determine the correct animal biosafety levels for your laboratory.

Q57. I work in a laboratory room that is shared with other labs, but I don’t think it is a core facility. Should I list it in the core facilities section or in the in vitro/in vivo use section(s)?

List work with biohazards in shared laboratories in the in vitro and/or in vivo rooms questions. If you are unsure if a laboratory is a core facility, reach out to EH&S Biological Safety at ehsbio@uw.edu or the biosafety officer assigned to review your BUA application. The biosafety safety office maintains a list of facilities considered core facilities. These sites are regularly inspected outside of the review cycle of individual projects. These facilities include the UW Greenhouse, Flow Cytometry cores, the stem cell core, MRI facilities, etc.

Q89. Why should I take the biosafety training?

As mandated by the NIH Guidelines, PIs must complete the EH&S Biosafety Training prior to their initiation of work with biohazardous agents and every three years thereafter. The training is also required for students, fellows, laboratory managers, research staff, and other staff with potential for exposure to biohazardous agents, including rDNA, at the UW.

The training covers roles and responsibilities when conducting research with biohazardous agents, including rDNA. The course also covers the review and approval process for biohazardous agents at the UW and the requirements governing their use (e.g., facilities, equipment, practices).

It is recommended that you take the training before completing this application. Be aware that your application cannot be approved until completion of training is verified. 

Q91. I only ship/transport Category B materials. Do I still need to take the Shipping Hazardous Materials Training?

If you are only shipping Category B materials, then no, the Shipping Hazardous Materials training is not required. However, if you work with more than just Category B materials or would like to learn more about the different classes of hazardous materials and their requirements, then the Shipping Hazardous Materials training is recommended.

 The Shipping Biological Substances Category B course meets the training requirements for shipping materials classified as a Biological substance, Category B, in the Department of Transportation (DOT) and International Air Transportation Association (IATA) regulations. The training includes instructions for packaging, labeling, and preparing shipping papers and is required to be taken every two years if you are actively shipping such materials.