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Risk Assessment made under the Genetically Modified Organisms (Contained Use) Regulations 2000 (Form GMM – for genetically modified micro-organisms and eukaryotic cell and tissue culture systems) Department: Supervisor: Ref. No: Project Title: Molecular retrovirology and cell-cell spread of retroviruses Overview of Project: (include aims and objectives) The aim of the project is to characterise the molecular events that facilitate efficient assembly of retroviruses such as HIV-1 and direct cell-cell spread of virions across the virological synapse. We have shown previously that HIV-1 assembly in infected cells and entry into target T cells requires the cellular actin and microtubule cytoskeleton as well as elements of the cellular secretory pathway, In order to fully dissect the involvement of these, and other as yet undefined, cellular compartments in HIV-1 infection it is becoming necessary to individually examine the contribution of specific cellular proteins in HIV pathogenesis and cellcell spread. Moreover, in order to monitor the transport of HIV-1 proteins individually, in the simplest system possible, and to assess the influence of HIV-1 proteins on global cellular trafficking events we wish to examine recombinant HIV-1 proteins expressed individually in cell lines to mimic key events in HIV infection. To achieve these aims we will use recombinant molecular biology and cell culture techniques that are routinely performed in labs all over the world. These include PCR amplification and cloning of genes of interest into commercial bacterial and mammalian expression vectors. Genes encoded by these vectors can then be introduced into mammalian cell lines by transfection technology (for example cationic lipid transfection or nucleofection) to generate stably or transiently modified cell lines for subsequent use. Give details of Recipient/Host(s): Vector(s): (specify if wild type or disabled) Standard bacteria vectors and mammalian expression vectors (eg pUC18 and pEGFP etc) Disabled E. coli, K12 and B derivatives, and BL21 and similar Mammalian cell lines (eg HeLa 293T, T cells) Normal/expected biological action of inserted DNA/RNA or transcribed/translated gene product: DNA encoding individual HIV-1 proteins and their derivatives (eg. HIV Gag) and DNA encoding human DNA and their derivatives (eg. CD4, CXCR4, Arp2/3) will be prepared and transfected into mammalian cell lines (eg HeLa, T cells) and primary human T cells to yield transiently or stably modified cell lines Technique used to introduce insert or vector into host: Genes are inserted into vectors by PCR amplification and restriction enzymes digestion and ligation or by subcloning. Vectors will be introduced into mammalian cells by standard transfection methods (eg. Lipofectamine, nucleofection) Assessed By: Signature: Date: Risk Assessment approved by Genetic Modification Safety Committee Signature: Date: (Biological Safety Officer) 1 Permission granted by Head of Department for project to be undertaken Signature: Date (Head of Department) 2 RISK ASSESSMENT FOR HUMAN HEALTH AND SAFETY Human health hazard identification – (Identify any potential harmful properties of:) i) the recipient micro-organism(for microorganisms also give ACDP hazard group) ACDP1 for all bacterial recipients. E.coli strains are disabled and cannot colonise the human gut. Minimal hazard for murine and human cell lines obtained from commercial sources that are well characterised and authenticated – containment level 1. Primary human cells and cell lines that are not fully authenticated and characterised may carry contaminating infectious agents – containment level 2 required under the COSHH Regulations. PCR amplification from replication defective HIV DNA plasmids does not pose a health concern as the constructs do not encode a complete functional HIV genome and so the DNA cannot produce infectious virus. PCR amplification from plasmids encoding full length infectious clones of HIV will be performed in the Containment Level 3 lab and following Containment Level 3 safety protocol means the risk is negligible. ii) the inserted (donated) genetic material Inserts code for normal mammalian genes or selective alterations of those genes. Also standard marker genes such as lac Z, GFP, etc. Inserts are not expected to have harmful physiological or pharmacological properties or to affect pathogenicity of cloning host or normal human defence mechanisms. Inserts will also code for individual HIV genes. Individual HIV-1 genes cannot generate virions and there is no risk of infection with these constructs. Gene transfer is possible but unlikely to be hazardous. Recombination or complementation with additional HIV-1 proteins is not possible in the context of current use. iii) the donor micro-organisms (where used/appropriate) The inserts are from mammalian sources and from HIV-1 iv) the vector Non-hazardous standard plasmid or mammalian expression vector systems. v) the resulting genetically modified micro-organism No significant hazards identified above. The HIV-1 proteins expressed are not infectious and when expressed individually are incapable of producing virus. The resulting GMOs are therefore not expected to carry any additional risks to that of the un-modified recipients. Brenner Scheme values (COMPLETION OPTIONAL and in any case for disabled E. coli only) Access Expression Damage Overall 3 Control measures – Assign provisional containment level: Containment Level: 1 (2 and 3 required under COSHH Regulations) with Good Microbiological Practice and Good Occupational Safety and Hygiene Note: under COSHH Regulations some cell lines require Containment Level 2 plus microbiological safety cabinet NATURE OF WORK TO BE UNDERTAKEN All standard molecular biology laboratory procedures such as PCR, restriction enzyme digestions, agarose gel electrophoresis Techniques include gel electrophoresis, blots, sequencing, FACS analysis, standard biochemical assays, immunocytochemistry, protein binding studies Max volume of cultures: up to 1 litre (multiples of) None Additional control measures required for specific risks: None 4 RISK ASSESSMENT FOR ENVIRONMENTAL HARM GUIDANCE Environmental hazard identification - Identify any potentially harmful properties of: Potentially harmful effects include: disease to animals including allergenic and toxic effects i) the recipient micro-organism None. No disease or other harmful effects to humans, other animals or plants. ii) the inserted (donated) genetic material None for inserts code for normal mammalian genes, or selective alterations of those genes. Also standard marker genes such as lac Z, GFP, etc. Inserts encoding individual HIV-1 genes do not pose any additional risk. Inserts are not expected to have harmful physiological or pharmacological properties or to affect pathogenicity of cloning host. iii) iv) the vector None. Majority are standard commercially available vectors with no harmful effects. vi) the resulting genetically modified micro-organism None. Resulting GMOs carry no additional hazards compared with those already present in the environment. Any transfer of genetic material to other organisms would be of minimal hazard. GMOs would not survive outside laboratory conditions. E.coli strains do not colonise the human gut; mammalian cells will not survive outside laboratory conditions. Where potentially harmful effects are identified estimate: Negligible i) consequence/severity of effects: ii) likelihood of effects being realised (taking containment and control measures assigned above into account): Negligible iii) overall risk: Effectively zero Additional control measures required to reduce all risks to low/effectively zero: None CLASSIFICATION AND ASSIGNMENT OF FINAL CONTROL MEASURES Consider each item on Table 1a indicate whether or not it is required taking account of the provisional containment level assigned to protect human health and safety and any additional control measures necessary to control specific activities and environment risks Consider also Tables 1b and 1c where appropriate See Table 1a 5 adverse effects resulting from establishment or dissemination of the GMMs in the environment adverse effects resulting from the natural transfer of inserted genetic material to other organisms the donor micro-organisms (where used/appropriate) Inserts encoding individual HIV-1 genes do not pose any additional risk. v) disease to animals and plants adverse effects resulting from inability to treat disease or offer effective prophylaxis Classification: Class: 1 Assign corresponding level of containment: Containment Level: 1 (2 and 3 required under COSHH Regulations) specify any other control measures required Note: under COSHH Regulations some cell lines require Containment Level 2 plus microbiological safety cabinet. 6 Table 1a: Containment Measures for Activities involving GMMs in Laboratories Where an item is listed as "may be required" this indicates the item to be an option at that particular containment level and its requirement should be determined by the risk assessment for the particular activity concerned. Delete no or yes as indicated by risk assessment. Containment Measures Containment Levels 1 2 3 4 Isolated laboratory suite not required not required required required Laboratory sealable for fumigation not required not required required required Surfaces impervious, resistant and easy to clean required for bench required for bench required for bench and floor required for bench, floor, ceiling and walls Entry to lab via airlock not required not required may be required no / yes required Negative pressure relative to the pressure of the immediate surroundings not required may be required no / yes required required HEPA filtered extract and input air not required not required required for extract required for input and extract Microbiological safety cabinet/enclosure not required may be required no / yes required required (class 3) Autoclave required on site required in the building required in the lab suite required in lab (double ended) Access restricted to authorised personnel not required required required required Specified measures to control aerosol dissemination not required required so as to minimise required so as to prevent required so as to prevent Shower not required not required may be required no / yes required Protective clothing suitable protective clothing required suitable protective clothing required suitable protective clothing required complete change of clothing and footwear Gloves not required yes required required Control of disease vectors (eg rodents, insects) which could disseminate GMMs no required required required Specified disinfection procedures in place yes required required required Inactivation of GMMs in effluent from handwashing sinks, showers etc not required not required may be required no / yes required Inactivation of GMMs in contaminated material and waste required by validated means required by validated means required by validated means required by validated means Laboratory to contain its own equipment not required not required required required An observation window or alternative so that occupants can be seen no may be required no / yes required required Safe storage of GMMs no required required secure storage required Written records of staff training not required may be required no / yes required required CLASSIFICATION CLASS 1 CLASS 2 CLASS 3 CLASS 4 7