Download Department:

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
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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
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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.
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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
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