What goes into my Biological Inventory?

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1 What goes into my Biological Inventory? What Information is Required for an Effective Risk Assessment? According to the Laboratory Biosafety Guidelines (2004), the risk group of an organism is determined by the following factors: Pathogenicity Infectious dose Mode of transmission Host range Availability of effective preventative measures Availability of effective treatment Please submit suggestions for clarification to A risk assessment is then done to determine the containment level (biosafety level) required to handle that organism. Your procedures and protocols are required for this portion. This risk assessment takes into consideration the following additional factors: Potential for aerosol generation Quantity Concentration Agent stability in the environment (inherent biological decay rate) Type of work proposed (e.g. in vitro, in vivo, aerosol challenge studies) Additionally, the widespread use of recombinant organisms as gene transfer vectors or other tools requires the additional risk group factors to be considered: Oncogenicity (addition of oncogene or deletion of tumor suppressor) genes coding for virulence factors or toxins host range alteration replication capacity capability to revert to wildtype Human and/or Animal Pathogens Here we refer to any virus, bacteria, fungus, toxin, protozoan, cultured eukaryotic or prokaryotic cells, cultured tissues of any species, cells cultured from blood or body fluids of any species, blood or body fluids from human or non-human primates. All materials listed above must be included in the inventory. The materials listed above may also have multiple variants which fall into one of the below categories which further clarifies the detail which is expected in the inventory. Approved August 27, 2012 Page 1 of 5

2 Modified Organisms vs Recombinants There are two general ways in which organisms are used in a research setting. The first is where the organisms itself is studied; it is the model, as when a virologist studies a virus. When the virologist modifies the wildtype to ascertain virulence factors or to study pathogen-host interactions, we refer to these organisms as modified organisms. The second is where the organisms are used purely as tools for gene manipulation, expression or transfer. We refer to these latter organisms as vectors or parent vectors. The parent vector may be your control vector or empty vector. Any further modification of the parent vector will be referred to as the recombinants. Please note that we are not referring to host organisms which carry plasmids or other types of nucleic acid sequences. Those organisms are addressed in the Libraries and Collections. In addition, a library can also be composed of recombinants. Those organisms are also covered in the Libraries and Collections section. Wildtype = the genotype and phenotype of the pathogen as it is found in nature. Wildtypes must be included in the inventory. Modified Organism = the wildtype organism has been modified in such a way that its properties may be studied in isolation or its pathogenicity may be studied inside a host. If the modified organism shows any changes in host range, oncogenicity (overexpress an oncogene or silence a tumor suppressor), virulence, replication capacity compared to the wildtype, then it must be listed in the inventory. Parent Vector = the modifications made to the wildtype pathogen (typically many sequences of DNA removed, often referred to as safety features by commercial suppliers). Parent vectors must be included in the inventory. Recombinants = the subsequent modifications made to the parent vector (typically insertion of a DNA sequence or modified DNA sequence). If an oncogene is to be overexpressed, that recombinant must be listed in the inventory. If a tumor suppressor is to be silenced or deleted, that recombinant must be listed in the inventory. If the recombinant shows any change in host range, oncogenicity, virulence, replication capacity compared to its unmodified parent, a risk assessment must be done. It is imperative to be aware that your intended modification may also result in some unexpected phenotypes. If none of the above applies, the recombinant does not need to be listed in the inventory. To be clear, the transgene may afford some pathologies to the test animal, however the basic properties of the vector have likely not been changed. Libraries and Collections Bacterial, yeast, baculovirus, bacterial artificial chromosome, yeast artificial chromosome Approved August 27, 2012 Page 2 of 5

3 A library is set of unknown nucleic acid sequences, which may or may not be expressed, hosted in an organism which a user screens for their target molecule. A collection is a set of known nucleic acid sequences, which may or may not be expressed, hosted in an organism which a user screens for their target molecule. Genomic library or collection = sequences of exonic, intronic and intergenic DNA, not expressed by host machinery. The host organism of the library or collection must be listed in your inventory. cdna library or collection = these may fall into one of three categories 1. sequences of reverse-transcribed messenger RNA which exist solely within plasmids in a host which CANNOT express the proteins (e.g. in bacteria such as DH5α whose purpose is NOT to express) 2. sequences of reverse-transcribed messenger RNA which exists as plasmids within a host which CAN express the proteins (e.g. in bacteria such as BL21 and induced by IPTG) 3. sequences of reverse transcribed messenger RNA which are integrated into the host genome (e.g. in lentivirus or bacteriophage) and are expressed when cells are transduced For cases 1 and 2, only the host organism is required to be in the inventory for both libraries and known collections. If commercial, please indicate company and product number. In the case of lentivirus-based expression libraries, it is possible that an oncogene is present as the contents of the library are unknown. By the same token, in the case of a lentivirus-based shrna library, it is possible that a tumor suppressor may be silenced since the contents of the library are unknown. Lentiviral libraries where the population of sequences to be expressed are UNKOWN are to be described in the inventory. Lentiviral recombinants where the sequences to be expressed ARE known, requires only those that encode for oncogenes, shrnas for tumor suppressors, virulence factors, toxins, for replication factors, altered trophic factors or enough homologous sequences such that recombination events and reversion to wildtype are likely, to be listed in the inventory. Transient Transfectants Here we refer to eukaryotic cells that have had exogenous DNA introduced which is NOT integrated into the host genome. The DNA is considered to be ectopic or extra-chromosomal. This exogenous DNA is transcribed and translated. It is NOT replicated. The parent cell must be included in the inventory. The transient transfectant does not. Stable Transfectants Here we refer to eukaryotic cells that have had exogenous DNA introduced which IS integrated into the host genome. The integration may be homologous or non-homologous. This exogenous DNA is replicated, transcribed and translated. Approved August 27, 2012 Page 3 of 5

4 The parent cell must be included in the library. If it has altered risk group factors compared to its parent, then the stable transfectant must be included in the inventory. If the risk group factors have not changed, then the stable transfectant does not need to be included in the inventory. Cell Line Panels Here we refer to mammalian cell lines, purchased as a collection of different tissues / cancers for use in screening. Cell lines must be included in the inventory. The principal investigator should supply the product information. Please enquire before starting data entry as the biosafety office may already have this information on file. McMaster-Sourced Untreated Animal Tissues Here we refer to tissues harvested from untreated sacrificed animals or blood or other body fluids harvested from untreated sacrificed or live animals that have been bred and maintained or purchased via McMaster s Central Animal Facility authority and are in a Sentinel Monitoring Program. Animal tissues as listed above do not need to be listed in the inventory if they are FIXED (as with paraformaldehyde, glutaraldehyde, neutral buffered formalin) PRIOR to exiting the animal facility. Animal tissues as listed above do not need to be listed in the inventory if they are to be PROCESSED (RNA / DNA / Protein extraction) within the laboratory. Animal tissues as listed above DO need to be listed in the inventory if they are to be maintained and or propagated in vitro. Tissues derived from ANY animal infected with ANY pathogen must be included in the inventory and handled at the containment level of the pathogen. Please note that any fixative used to fix tissues from PATHOGEN EXPOSED animals, must also be effective against that same pathogen. If the fixative is not proven effective against that same pathogen, the tissue must still be handled at the containment level assessed for that pathogen. Lentiviral Plasmid Systems Here we acknowledge that these exist as multi-plasmid based systems. Plasmids in and of themselves are BSL1, however once created, the lentiviral particle is a minimum of BSL2. The plasmids may be submitted as an attachment, or product datasheets may be submitted. Typically a plasmid system consists of a number of plasmids which are NOT altered. These plasmids contain sequences which are transcribed and translated into proteins. The mrna transcribed from these plasmids do NOT contain the psi sequence. The one plasmid that DOES contain the psi sequence also typically contains the gene of interest (GOI). If the system is commercial, the system name can be used for identification. If non-commercial, the psi/goi plasmid can be listed as the system, the other plasmids may be described under additional Approved August 27, 2012 Page 4 of 5

5 information. The information to provide are things such as # plasmids, pseudotype, viral proteins to be produced, viral species upon which the system is based etc. The variants of each psi/goi plasmid must be listed in the inventory if the GOI encodes for oncogenes, sirnas for tumor suppressors, virulence factors, toxins, for replication factors, altered trophic factors or enough homologous sequences such that recombination events and reversion to wildtype are likely. Otherwise only the original psi/goi plasmid need be listed in the inventory. If you use different psi/goi plasmids with the same packaging plasmids, that different psi/goi plasmid must also be listed in the inventory. The cells which are used in the system must be listed in the cells inventory. For lentivirus-based libraries, see above section Libraries. The library is to be handled at BSL2+. Any individual clone isolated from the library must have its own risk assessment to ascertain its containment level. If You are Unsure If you are unsure if the modifications to your organism have any impact on the above referenced risk factors, please contact the biosafety office so we may conduct a risk assessment to ensure work is undertaken safely. Intent The Presidential Biosafety Advisory Committee relies on self-reporting on behalf of the principal investigator and laboratory audits. Should any change in phenotype be noted in a recombinant or other material listed above, the principal investigator is required to inform the Biosafety Office of these changes in order to conduct a detailed risk assessment. Going from BSL2 to BSL2+ In the event a risk assessment deems an organism or specimen be used under containment level 2+, a few minor changes in operational procedures are required. These changes are not difficult to implement and require the creation of additional SOPs. Please contact us for consultation on how these changes may be achieved in your laboratory. Approved August 27, 2012 Page 5 of 5