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Recombinant or Synthetic Nucleic Acid Molecules (r/sNA)

When reporting work with r/sNA molecules, please see the guidance below:
 

Gene Transfer Table (2a):

  • Name how the r/sNA molecule transfer into a cell is mediated/promoted (not the plasmids used). List all gene transfer methods as separate line items. If your Host-Vector System is replication competent, please include it in the “Infectious Agents” section also.  Include a gene transfer method to account for pre-made genetically engineered agents (unless of minimal risk) even if no gene transfer events may be occurring.  See the “Experiments Involving Pre-made Engineered Agents” section below.
    • Options for gene transfer methods are: physical, host-vector system, or other.
      • Physical method examples:
        • Microinjection
        • Electroporation
        • Heat shock
      • Host-Vector System examples:
        • Adenoviral transduction
        • Lentiviral transduction
        • Agrobacterium tumefaciens transduction
      • Other method examples:
        • Nanoparticles
        • Bacterial conjugation
        • Liposomal transfection
      • Complete the pop-out window for each transfer method.
        • Describe the gene transfer method and its biosafety features.
        • Include the strain of E. coli used for any related transformations.
        • If you are seeking biosafety level containment downgrading, check “yes” and provide a justification for the downgrade.
      • If lentiviral work will be done and BSL2 containment is desired (lentivirus is a risk group 3 agent), then you must provide a justification for the downgrade.  Include the generation of the packaging system and the packaging plasmid names, kit name (if relevant) in the justification for the downgrade and the transgenes that will be used.  If the lentivirus will express oncogenes or knockdown/out expression of anti-oncogenes, additional required PPE (including mask, safety glasses, and sleeves) must be stated in your SOPs for this work.

To help the IBC with their review, please include vector maps in the “Attachments” section which show the specifics (restriction sites, promoters, open reading frames, antibiotic resistance genes, selectable markers, etc.) of the plasmids used for r/sNA molecule experiments. Construct-specific vector maps are not necessary, just the background plasmids that are used for the expression of your r/sNA molecules of interest.

Importantly, please attach maps for all plasmids used in the generation of your viral vectors (generated in your lab, a collaborators, or a company/core facility). The IBC aims to verify the generation of your lentiviruses. And, the IBC must be able to determine the origins of the components used and the host range of your recombinant agents.”

 

Genetic Material to be Transferred Table (2b):

The genetic material requested is meant to account for the expression products outside of host-vector transfer systems (which, for example, if viral, are transferring viral genes).  This is also meant to account for areas/genes of recombinant infectious agents that will be modified.  The IBC (mostly) does not grant blanket approvals and requests specifics.  However, the IBC does understand this cannot be fulfilled with some discovery type experiments (IE, high-throughput mutagenesis). In such cases, the IBC request PIs do their best to be as specific as possible for the biosafety risk assessment.

Please note that the pop-out window for Table 2b (when you add genetic material) includes slightly different titles (column headers) than the default table in the application (we couldn’t change these).  The titles in the pop-out window are intended to cover a broader range of expression products (which is what the IBC wants).  If the title/request doesn’t fit what you are doing, use the field for information that may help with the biosafety risk assessment or write “NA” (not applicable).

  • Name of gene, cDNA, or RNA (Describe/Name r/sNA Molecule):
    • Add the names (or whatever designation you use) of the genes, DNA, RNA (not the plasmid names), or other r/sNA molecule to be transferred individually as separate line items.  If you use an abbreviation, please make sure you write out what it is in the “expression product” area unless this is a common/recognized gene abbreviation.
    • If there are many items to add (>10), an excel spreadsheet can be included in the attachments section.
    • Include Cas9 in the list for CRISPR work.
    • Add gene targets for gRNAs, siRNAs, shRNAs as separate line items.
    • Include any reporters (i.e. GFP or RFP) that are present in any plasmids used.
    • Describe all components of any fusion proteins as separate line items in the table.
  • Species or Origin:
    • List the species or origin of the gene of interest.
    • If a fusion protein is being used, list the species or origin of each component of the fusion protein.
    • If species or origin does not apply to the r/sNA molecule, write “NA”
  • Function of the Gene (or r/sNA Molecule):
    • List the general function of the gene or r/sNA molecule that is being transferred. For example: reporter, kinase, transcription factor, target gene, silence gene, etc. 
    • It is important to denote oncogenes or genes that have the potential to be oncogenic if suppressed.
    • If what you are doing doesn’t fit the request, write “NA”
  • Protein to be Expressed (Expression Product):
    • Break fusion proteins into individual components.
    • Write the full name of the gene rather than acronyms unless they are commonly known such as GFP.
    • If there is no expression product, write “NA” or describe the change (IE, promoter mutation).
    • List the product that will be expressed after transfer.
  • If the transfer of genetic material will result in an over expression or knockout of a gene/protein, indicate this in the column for “Expression Product.”

 
Examples:

Plasmid name- pAAV-hSyn-EGFP (EGFP under the control of human synapsin promoter)

Describe/Name r/sNA Molecule Species or Origin Function Expression Product
EGFP Jelly fish- Aequorea Victoria Fluorescent reporter EGFP

 

Plasmid name: pGP-CMV-GCaMP6f (mammalian expression vector containing a fusion protein calcium sensor)

Describe/Name r/sNA Molecule Species or Origin Function Expression Product
GFP Jelly fish Fluorescent reporter GFP- in fusion protein GCaMP6f 
CaM Human Calcium binding Calmodulin- in fusion protein GCaMP6f
M13 Human Targeting M13 peptide- in fusion protein GCaMP6f

 

siRNA to Human myosin light chain 1

Describe/Name r/sNA Molecule Species or Origin Function Expression Product
siRNA to MYL1 NA Knockdown of MYL1 siRNA to myosin light chain 1; knockdown of MLY1

 

Experiments Involving Pre-made Engineered Agents

Pre-made Engineered Organisms/Cells/Agents

Experiments which involve the use of pre-made genetically engineered or modified agents (e.g. organisms/cells/viruses/bacteria/etc) should be accounted for by including a gene transfer method in the Gene Transfer Table 2a, even though the agent is already recombinant and is not transferring r/sNA molecules (we are trying to get this header changed). We have eliminated the minimal risk form and procedure to consolidate this activity into one process. 

Examples of pre-made engineered agents:

  • Pre-made recombinant infectious agents (IE Salmonella with specific gene knockouts).
  • Pre-made viral-based vectors that are not transferring any r/sNA molecules to cells, but do contain r/sNA molecules.
  • Pre-made genetically engineered cell lines expressing a replication competent virus or biological toxin.  We do request some redundancy of information for some agents. For example, replication competent infectious agents (viruses, bacteria, etc) also need to be accounted for in the Infectious Agents section.  Expressed biological toxins also need to be accounted for in the Biological Toxins section of the application (if they meet the definition).
  • Pre-made recombinant agents which could impact the environment but are not necessarily infectious to humans (i.e., recombinant arthropod pests, recombinant noxious weeds, etc).
  • A human cell line that has been altered to express GFP.
  • A transgenic strain of drosophila that has been genetically altered by a collaborator to express a siRNA.
  • A pre-generated transgenic strain of mice expressing Cre.

To account for the use of purchased, provided, or already made genetically engineered or modified animals, please see the GM Animals section (6.).

To account for the use of other purchased, provided, or already made genetically engineered or modified agents:

  1. Select the box for “recombinant or synthetic nucleic acid molecules” in the “Activities Include” section.
    Complete the “Study Objectives” section (1) a and b, with a brief description and the goals/aims of the work involving the pre-engineered agents/organisms.
  2. In the “Recombinant/Synthetic Nucleic Acid Molecules (r/sNA Mol)” section (2):
  • Include a gene transfer method in Table 2a.  Select an “other” method of transfer and in the pop-up window section a) name the GM or GE infectious agent(s). In c), briefly describe the GM or GE agent(s) and source. In d), you will need to enter some words in order to fulfill an application dependency.
  • Include (as best you can for the risk assessment) the genetic material that the engineered agent/organism expresses or has altered in the Genetic Material to be Transferred Table 2b.  If biological toxins or replication competent viruses are expressed, also include this information in the Biological Toxin section (3) and/or Infectious Agent section (4) as appropriate.
  • In the “Safety” section (7) include:
    • The laboratory location where this work will be performed and where the GM/GE agent is stored with the proposed BSL in section 7a)i
    • Fill in sections b through h accordingly.
    • Under i) Laboratory Procedures, add the experimental activity and appropriate SOP title for the work with the pre-made agent/organism.
  • In the “Attachments” section attach:
    • Biological Decontamination and Spill Clean-up Plan (tailor to your lab and application)
    • Biological Waste Disposal Plan (tailor to your lab and application)
    • Corresponding vector maps (i.e, lentivirus maps including the packaging vectors maps). Note: An attachment must be included as a vector map if r/sNA work is indicated in the application.  If there is no vector map, please attach a document that states, “no vector map for pre-made agent.”
    • SOPs for the use and handling of the pre-made engineered agent/organism.

     

    Transformations of K-12 strains of E. coli or use of recombinant K-12 strains of E. coli

    Although exempt from NIH Guidelines (III-F), transforming E. coli K-12 strains or culturing recombinant K-12 E. coli must be approved by the IBC. IBC applications that include only this work can typically undergo an expedited review. Recombinant protein expression typically uses E.coli that is not K-12 and is not exempt from the NIH Guidelines. Use of risk group 3 or 4 genes/sequences, or sequences that produce toxins require a full IBC review.
     

    To add the transformation of E. coli (K-12 strains) to an IBC application:

    1. In the “Activities Include” section, select “Recombinant or Synthetic Nucleic Acid Molecules.” 
    2. Complete the “Study Objectives” section (1) a and b, with a brief description, goals/aims of this exempt work.
    3. In the “Recombinant/Synthetic Nucleic Acid Molecules (r/sNA Mol)” section (2):
    • Add a gene transfer method (chemical/physical) and fill in the corresponding pop-out window. Some methods typically used are heat-shock or electroporation of chemically competent E. coli. Include the strain of E. coli used in the description section (a) of the pop-out window (non-K-12 strains are not exempt).
    • List, as separate line items, all r/sNA molecules (genes/sequences) under “Genetic Material to be Transferred” (b). (typically not the backbone vector genes/sequences unless they will be expressed in eukaryotes)
  • In the “Safety” section (7) include:
    • The laboratory where this work will be performed (a-i).
    • Fill in b through h accordingly.
    • Under i) Laboratory Procedures, add the experimental activity and appropriate SOP title.
  • In the “Attachments” section attach:

    • Follow the above steps to add the transformation of E. coli (K-12 strains) to an existing application with multiple IBC oversight activities or to an application where the transformation of E. coli K-12 strains is the only IBC related activity.

    To add the culturing of recombinant K-12 strains of E. coli to an IBC application (this is not necessary if adding transformation to your application – and following the above procedure):

    1. In the “Activities Include” section, select “In Vitro” and “Other” and note “recombinant K-12 strains of E. coli.”
    2. Complete the “Study Objectives” section (1) a and b, with a brief description, goals/aims for the culturing of K-12 strains of E. coli in your laboratory.
    3. In the “Safety” section (7) include:
    • The laboratory where this work will be performed (a-i).
    • Fill in b through h accordingly.
    • Under i) Laboratory Procedures, add the experimental activity and SOP title.
  • In the “Attachments” section attach:

    • Follow the above steps to add the culturing of recombinant E. coli (K-12 strains) to an existing application with multiple IBC oversight activities or to an application where culturing of recombinant E. coli K-12 strains is the only IBC related activity.

    NIH Guidelines Designation (2c)

    Please designate the section(s) of the NIH Guidelines for Research Involving Recombinant DNA Molecules under which your experiments are covered (e.g., III-D-1-a, III-E, etc.). Final designations will be determined by the IBC. 

    • Commonly used designations:
      • III-D-1-b for lentiviral transduction
      • III-D-1-a for adenovirus transduction
      • III-D-2-a for the expression of DNA from RG2 organisms in E. coli (such as Cas-9)
      • III-E for transfections, AAV transduction, or transformations of non K-12 strains of E. coli
      • III-F for the transformation of K-12 strains of E. coli or breeding/use of transgenic mice

    The University of Minnesota Board of Regents Policy does not characterize activities subject to IBC oversight that are specified in the NIH Guidelines as non-exempt or exempt.

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