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The document provides an overview of genetically modified organisms (GMOs) including what constitutes a GMO, procedures that do not result in GMOs, key duties regarding contained use of GMOs, risks associated with GMOs, classification of GMOs, and what to do in the event of a spill in a containment level 2 laboratory. It defines GMOs and discusses bacterial expression systems used to produce proteins of interest from gene inserts. It also outlines the risk assessment approach and regulations for working with GMOs.
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- 1. Foundation Training inBiological Safety
- 2. Module 4 Geneticallymodified organisms
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- 5. What is GM?(e.g. bacterial expression system) Page Gene of interest (insert) Vector Host Protein of interest
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- 8. Procedures not considered as GM Mutagenesis- chemical or radiation Cell fusion of prokaryotic species which can exchange genetic material through homologous recombination Cell fusion of cells of any eukaryotic species, including production of hybridomas Self-cloning, where the resulting organism is unlikely to cause disease or harm to humans, animals or plants Page
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- 11. The GMO (Containeduse) Regulations: key duties Assess risks created by work to either human health or the environment presented by GMMs/GMOs Review and record risk assessments Notification of Class 2 and 3 GMM activities Apply the principles of occupational and environmental safety Apply the containment measures specified Have in place emergency plans Report accidents
- 12. GM Regulations WhereGMMs or GMOs are cultured, stored, used, transported, destroyed or disposed of. Contained using barriers to protect the general population and environment Physical Chemical Biological Management Page
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- 14. Example of GM(bacterial expression system) Page Gene of interest (insert) Vector Host Protein of interest
- 15. Risks HOST Howpathogenic? Virulence and infectivity? Are toxins produced? Allergenic factors? Seriousness of exposure? Survivability outside lab? Page
- 16. E.coli K12, the prokaryotic wimp
- 17. RISKS VECTORSConsider: Pathogenicity Transferability of the vector: Only want vector to transfer target host, achieved by modifying how a vector transfers Page
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- 19. RISKS INSERTS Consider: gene expression e.g. overexpression hazards posed by the gene product e.g. activity of expressed protein, toxic, allergenic or pathogenic effects Different effects depending on the location of the insert Alteration of existing pathogenic traits? Page
- 20. THE GM (CONTAINEDUSE) REGULATIONS Requires that all GMMs are classified in one of four Classes Class 1 Class 2 Class 3 Class 4 Increasing hazard to human health or the environment 1 2 3 4 Containment Level Classification of GMM determined by Containment Level required
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- 22. GM animals orplants Genetic material altered using a method that does not occur naturally. Not chemical or physical mutagenesis Stable integration of the inserted GMM into the genome Direct injection of naked DNA to elicit an immune response not GM Page
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- 25. RISK ASSESSMENT APPROACHFOR GM ANIMALS AND PLANTS Harmful nature of GMO Environmental Human Health Page
- 26. The GMO (Containeduse) Regulations Requires that all GMOs (other than GMMs) are classified on the basis of whether or not they are HSE notifiable GMO poses no risk to man greater than that of the parental organism GMO poses a greater risk to man than that of the parental organism HSE notifiable
- 27. Keep paperwork up-to-dateReview when you make a change How? GM Form C Page
- 28. GM WASTE Inactivateby validated means Method must work under the conditions of use Chemical disinfection/autoclaving Page
- 29. INFORMATION SOURCES HSEWebsite: http://www.hse.gov.uk/hthdir/noframes/hthdir.htm SACGM Website: http://www.hse.gov.uk/biosafety/gmo/acgm/acgmcomp/index.htm ACDP Website: http://www.hse.gov.uk/hthdir/noframes/agent1.pdf GM Regs: http://www.hmso.gov.uk/si/si2000/20002831.htm Safety Department Website: http://www3.imperial.ac.uk/safety/guidanceandadvice/biosafety Page
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- 32. Workshop Scenario Youenter a Containment level 2 lab to retrieve your bacterial cultures that have been growing overnight in a shaking incubator. Upon entering the lab you find that there has been a spillage of an unknown culture from one of the incubators onto the floor. What do you do?
- 33. Model Answer Switchoff incubator Ascertain what was in the culture by referring to the labelling system or log book Contact relevant researcher and local safety officer Refer to spillage SOP Wear appropriate PPE Use disinfectants at the specified conc and contact time. Ensure safe disposal of contaminated material. Complete College DO form If Class 2 GM material Safety Dept must notify HSE. Further investigations required.
Editor's Notes
- #4 GM makes it poss to manipulate organisms to be safer, more specific,more productive in what they do compared to their unmodified counterparts. This technology produces novel organisms and they may affect human health and interact with the environ in new and possibly harmful ways therefore have regs governing this work. Optional slide
- #6 Insert of interest into a virus, bacterial plasmid or other vector system into a host organism in which they do not naturally occur but in which they are capable of continued propagation. Direct introduction of heritable genetic material into an organism , including micro-injection and macro-injection. Cell fusion/hybridisation techniques where live cells with new combinations of heritable genetic material are formed through the fusion of two or more cells by artificial means This schematic illustrates simply how a GMM is made e.g. a bacterial expression system The gene of interest/insert is placed into a vector The vector and insert are then placed into a host And this final product is the genetically modified organism So the risk of the final GMM is not assessed alone but rather all the components that go into making the GMM and also the final product too
- #7 GFP visualisation of genetic modification - Two mitochondrial and two plastid-targeted PPR-GFP fusions in tobacco protoplasts
- #8 GFP visualisation of genetic modification - (mouse on the right is transfected with GFP)
- #9 Other techniques that the GM regs do not apply to include: Mutagenesis, this includes that induced by chemicals and radiation, but not site directed mutagenesis where recombinant nucleic acid molecules are used. Cell fusion where the cells are derived from prokaryotic species which are known to naturally inherit each others genomic DNA through homologous recombination and integration. Cell fusion of cells from eukaryotic species including the production of somatic animal hybridomas and the fusion of plant cells. Self cloning micro-organisms are exempt only if the resulting GMM is unlikely to cause disease or harm (allergenicity, toxicity and infection) to animals or plants. Self cloning is where all of the inserted nucleic acid involved is derived from the same species or one with which it can naturally exchange genetic material by homologous recombination.
- #10 IVF not a GM technique therefore exempt from the GM Regs.
- #11 RNAi Gene silencing by RNA interference (RNAi) is an antisense technology assessing gene function in mammalian cells. The two most critical factors determining the effectiveness of RNAi experiments are the ability of the siRNA sequence to specifically silence the target mRNA, and the efficiency with which siRNA (or an shRNA-expressing vector) can be transfected into the cells of interest. RNA interference (RNAi) is the process where the introduction of double stranded RNA into a cell inhibits gene expression in a sequence dependent fashion. RNAi is usually described as a post-transcriptional gene-silencing mechanism in which dsRNA triggers degradation of homologous messenger RNA in the cytoplasm. The mediators of RNA interference are 21- and 23-nucleotide small interfering RNAs (siRNA). In a second step, siRNAs bind to a ribonuclease complex called RNA-induced silencing complex (RISC) that guides the small dsRNAs to its homologous mRNA target5. Consequently, RISC cuts the mRNA approximately in the middle of the region paired with the antisense siRNA, after which the mRNA is further degraded. Where synthetic siRNAs are transfected into the cell = Not GM Delivery of siRNA via a vector (bacterial or viral) = GM activity
- #12 They main requirements that must be completed according to the GM regs are: The risk from GMMs or GMOs to human health and the environment are fully assessed That the risk assessments are recorded and reviewed The location where any GM work is carried out is notified to HSE If the GM work is Class 2 or higher, notification to HSE is required Establish a GM committee Ensure that GM work is controlled by the appropriate containment measures, occupational and environmental safety principles Adequate emergency plans in place and reporting procedure for all accidents that includes GM specific work
- #13 Definition of contained use relating to GM Any activity where a genetically modified organism is created or present Where that activity is controlled by barriers i.e. physical, chemical, biological. Type of controls used to limit contact will depend on the risk assessment Purpose of the GM Regs Stop or limit contact between GMM/GMOs and humans and the environment by the use of biological, physical and chemical barriers Barriers used are proportional with the risks associated with the GMMs/GMOs
- #14 RISK ASSESSMENT APPROACH Harmful properties of the GMM Potential to cause harm Environment that is likely to be exposed Characteristics of the activity Ensure appropriate containment and control measures are taken to reduce the likelihood of harm occurring Disposal options The risk assessment should include: The identification of any potentially harmful effects, specifically those associated with: the recipient organism the inserted genetic material the vector the donor organism the resulting GMO the characteristics of the activity involving genetic modification the severity of the potentially harmful effects, e.g. disease to humans including allergenic or toxic effects acting as a human disease vector or reservoir adverse effects to humans arising from change in behaviour or in physical nature adverse effects arising from the inability to treat human disease or offer effective prophylaxis the likelihood of these effects occurring
- #15 This schematic illustrates simply how a GMM is made e.g. a bacterial expression system The gene of interest/insert is placed into a vector The vector and insert are then placed into a host And this final product is the genetically modified organism So the risk of the final GMM is not assessed alone but rather all the components that go into making the GMM and also the final product too.
- #16 Risks associated with the Host Need to consider the host’s pathogenicity including the virulence, infectivity whether a toxin will be produced and whether any product the host produces could cause an allergic response What are the consequences of exposure, how serious could this be must be estimated. Can the host survive outside the lab environment? For example a low risk host choice would be E.coli K12 Disabled, avirulent, can only survive for a limited time outside the lab E.coli is a human pathogen which is assigned ACDP Hazard Group of 2 Explained earlier in the Biosafety training by Anton de Paiva So E.coli is HG2 however attentuated/ less virulent strains can be assigned a lower HG i.e. E.coli K12 therefore it lowers the containment level that needs to be used.
- #17 Survivability outside lab? E.g. E.coli is a human pathogen, ACDP hazard group 2. But attenuated E.coli K12 strain is disabled, so survival outside the lab is limited, therefore hazard group 1. Optional slide
- #18 Risks associated with the vector. There are two main things to consider: The pathogenicty of the vector- the majority of vectors used in E.g. bacterial vectors, majority of vectors used in E.coli do not contain sequences encoding pathogenic characteristics but viral vectors may retain some pathogenic characteristics The vectors ability to transfer to other organisms- if it non mobilisable, mobilisable deficient or self mobilisable the risk is eliminated (explain each) Non-mobilisable vectors - defective in one of more functions required for transfer , Mobilisation defective vectors - defective in one or more transfer functions, requires transfer function of another vector to help it transfer to target host Self mobilisable vectors - conjugative or can be mobilised by conjugative plasmids Viral vectors are becoming more commonly used. Very efficient but they can retain some of their pathogenicity In some cases cells can be infected but once inside they are replicant deficient Therefore they can deliver the DNA to the target cell but can’t replicate e.g. retrovirus based vectors Considerations: recombination with latent viruses in the host Insert affecting pathogenicity/tropism e.g. change of receptor recognition
- #20 Risks associated with inserts gene expression and the gene product…could the protein be toxic or have allergenic or pathogenic effects Location of the insert Could the insert alter the infectivity or pathogenicity Could a disabled host be enabled by the insert If accidental infection occurred is there any prophylatic treatment for the GMM? Treatment after an accidental infection, will the susceptibility of the GMM to antibiotics be affected due to the genetic modification? Could any disabling mutation within the host be overcome due to the insertion of the foreign gene? Mechanism of attenuation, deletion mutations are less likely to revert to wildtype than point mutations or conditional-lethal mutants. Consequence of reversion in an attenuated/disabled strain could be generation of a pathogenic strain that expresses the inserted gene. Minimised by placing insert at the site of an attenuating mutation, therefore any recombination event that restores previously deleted sequences will result in the deletion of the inserted gene.
- #21 Assess hazards associated with each. Indicates the risk associated with the recombinant organism and the appropriate lab containment level is assigned. Classification is directly related to the four levels of containment for microbial laboratories required to control the risk of the hazard to human health and the environment. Initial classification is based on the biological agents hazard group. Classification system based on the risk of the activity independent of the purpose of the activity.
- #22 Health Surveillace: Class 2 and above GM work should be discussed with OH
- #23 The GM Regulations cover the breeding on of a GM plant/animal. Even when a GM plant/animal is crossed with a non-genetically modified one, the progeny will be considered to be GMOs, unless tests can show that no modified genetic material has been passed to the progeny. GM Techniques include those which incorporate recombinant nucleic acid into the organism in a relatively stable form, even if not all cells of the organism contain the modification
- #24 ENU is one of the most effective alkylating mutagens and is known to randomly induce point mutations. Since most human genetic diseases are caused by partial loss of the gene function due to point mutations, mouse mutants obtained by ENU-mutagenesis are good animal models for human diseases.
- #25 By determining the function of genes on a mouse chromosome, you can extrapolate to predict function on a human chromosome. ENU mutagenesis chemically induces point mutations therefore these mice are not considered to be genetically modified.
- #26 Similar to GM microorganism except disease reservoir and behaviour considerations Environmental Invasiveness, toxic, disease reservoir, competition with wild type Human Health Allergenicity, toxic, disease reservoir, behaviour The consequences to the natural population should be considered if a transgenic animal were released accidentally or escaped from the laboratory
- #28 The risk assessment is a living document that requires review the assessment is no longer valid scope of project has changed significant change change of scale of the operation change in containment conditions change of waste treatment procedures new data on the behaviour of the organism e.g. change in scale operation i.e originally a 1L culture is now a 5L culture New data on behaviour of an organism e.g. lentiviral use was a Class 1 CL1 organism but new data revealed its tumorigenic properties and therefore all work with such viruses must be carried out at CL2 Document control procedures state that risk assessments must be kept for 10 years from the date that the work finishes
- #29 Chemical disinfection Difficult to validate Conditions may be variable Efficacy range Validation: Refer to manufacturers data if used as specified Concentration Contact time Exact conditions of use Autoclaving preferred British Standard BS EN 12740:1999 “Biotechnology - Laboratories for research, development and analysis - guidance for handling, inactivating and testing of waste” provides useful information on systems for dealing with contaminated waste. The inactivation method chosen should work and the details of the procedure specified so that it can be carried out in a reproducible way. Data regarding the method chosen can be obtained from different sources, such as: previous experience; laboratory studies; literature review. This data should indicate the inactivation conditions that are required. The chemical agent to be used must be fully active against the particular micro-organisms being used and the conditions of use should not reduce the activity of the chemical agent to an unacceptable level. Some disinfectants can be affected by the chemical environment in which they are used and their activity greatly reduced, due to this fact actual measurements should be taken to quantify the efficacy of the method being used. Validation results should be recorded along with the exact conditions of used. If the condition of use changes then the validation procedure should be repeated. Usually validation results are provided by the manufacturer and as long as the disinfectant is being used under the same conditions as specified in the manufacturer’s validation, this data is acceptable. Autoclaving is recommended as an alternative to chemical inactivation where validation of the chemical inactivation method proves difficult.
- #31 HSE website has information and guidance on GMOs SACGM is the Scientific Advisory Committee on GM They are the body responsible for advising the HSE on GM work They produce they produce the Compendium of Guidance Provides guidance for committee decisions on risk assessments for GM work