Develop* genetically modified organisms in containment · 2019. 4. 6. · Page 2 of 23 Please note...

ER-AF-N03-4 09/09

BP House

20 Customhouse Quay

PO Box 131, Wellington

Phone: 04-916 2426 Fax: 04-914 0433

Email: [email protected]

Web: www.ermannz.govt.nz

Application to

Develop* genetically modified organisms in containment Under section 40(1)(b) of the HSNO Act 1996 (excluding rapid assessment)

*“Develop” includes developing, fermenting and regenerating genetically modified organisms

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Please note

This application form covers the development of genetically modified organisms that:

1. Do not meet Category A and/or B experiments as defined in the HSNO (Low-

Risk Genetic Modification) Regulations 2003;

2. Occur either in a containment structure (i.e. laboratory) or outdoors within a

containment facility; or

3. Otherwise cannot undergo a rapid assessment for low-risk genetic

modification.

Any extra material that does not fit in the application form must be clearly labelled,

cross-referenced, and included as appendices to the application form.

Commercially sensitive information must be collated in a separate appendix. You

should justify why you consider the material commercially sensitive, and make sure it

is clearly labelled as such.

If technical terms are used in the application form, simply explain these terms in the

Glossary (Section 8 of this application form).

Unless otherwise indicated, all sections of this form must be completed for the

application to progress.

Applicants must sign the application form and enclose the correct application fee

(including GST). The application fee can be found in our published Schedule of Fees

and Charges on the ERMA New Zealand website. We are unable to process

applications that do not contain the correct application fee.

An electronic and paper copy of the final completed form must be submitted.

If you have any queries regarding the information required or would like to discuss

your draft application form, please contact a New Organisms Advisor at ERMA New

Zealand.

This form was approved by the Chief Executive of

ERMA New Zealand on 22 September 2009. This form replaces all previous versions.

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Section 1: Application details

a) Application title

Use of genetically modified zebrafish (Danio rerio) embryos and genetically modified Caenorhabditis

elegans at PC1

b) Organisation name

University of Auckland

c) Postal Address

Private Bag 92019

Auckland 1142

New Zealand

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Section 2: Summary of application

a) Provide a plain English summary of this application including:

Explain the purpose of your research in the context of your organisation’s history and goals.

The purpose of the application (e.g. what is the research you wish to perform and why do you consider that it is important? what are the benefits of this research?).

If there are any alternative methods to achieve the aims of this research, explain why you wish to perform the research this way.

Describe the project you wish to undertake (section 40(2)(a)(ii) of the HSNO Act).

Are you aware of any possible adverse effects of the organism on the environment? If so, any potential mitigation measures?

Where do you intend to conduct these activities? Are there specific location(s) or are you seeking approval for all of New Zealand?

How do other legislative requirements apply to your proposed activities? (e.g. the Resource Management Act, the Medicines Act.

If this application is for a development outdoors within a containment facility, discuss why your activities are not “field testing” activities for the purpose of the HSNO Act.

If technical terms are used here or elsewhere in the application, add simple explanations for these terms in the Glossary (Section 8 of this application form).

An exciting new collaboration involving microfluidics (see Appendix 1) to sort genetically modified

zebrafish (Danio rerio) embryos (less than 72 hours old) and Caenorhabditis elegans has prompted the review

of the containment level currently required to contain these very low risk organisms, in virtue of compliance

costs, and better directed methods to ensure containment.

Both organisms are classified as Category 2 host as per Regulation 7 (2) iii of the HSNO (Low Risk Genetic

Modification) Regulations, 2003. Category 2 hosts, and any modifications (genetic engineering) by default

is Category B, that require PC2 containment.

It is our opinion, that a there is a lack of clarity in the current standards for the containment of aquatic

organisms. Both zebrafish embryos and C. elegans have an absolute requirement for water for survival and

any stably integrated transgene, episomal transgene array (C.elegans) or stable gene knockout will not alter

this absolute requirement for water. We also propose that C. elegans and Danio rerio (embryos less than 72

hours old) with low risk modifications (i.e. qualifying as Category A modifications in Category 1 hosts) can

be easily contained within laboratories (including microfluidics laboratories) meeting the construction

requirements of PC1. We propose additional controls that specifically address potential pathway of escape

from containment of these organisms and thus, will deliver a superior containment outcome.

Furthermore the requirement for inward flow of air for the construction requirements of PC2, (and their

equivalent BSL 2 and ANSI laboratory standards) is driven by the containment of aerosols, which increases

compliance costs for no risk mitigation securing containment of either of these organisms. This application

requests that both of these organisms, including genetically modified organisms (as hosts with a proven

track record of laboratory safety), be contained in a dedicated facility approved to current the Standard for

microorganism at PC 1 with additional organism specific controls for their containment.

Zebrafish and C.elegans are commonly used models for the purpose of interrogating gene function. It is

relatively simple to knock down or knock out gene expression of specific genes using morpholinos

(synthetic molecules that are complementary to targeted bases) in the case of zebrafish or RNAi in the case

of C.elegans. In addition both species can be made transgenic through the injection of foreign DNA. This

DNA may code for specific genes of interest for instance; human disease genes or marker proteins such as

Green Fluorescent Protein (GFP) under the control of a tissue specific promoter. The functions of new

proteins can be determined by examining the resulting morphology of the organism and tissue for

molecular analysis. Both species can also be used for genetic screens identifying modifier loci via

mutagenesis and breeding or through knockdown library searches. Genetic modifier screens are often

undertaken on transgenic lines looking for potential drug targets. As part of a screening program drugs can

be tested directly on the organisms.

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b) Provide a short summary statement of the purpose of this application to be used on ERMA New Zealand’s public register

This statement must be a maximum of 255 characters including spaces and punctuation. If native or human genetic material directly obtained from New Zealanders is to be used, include this information here. Sufficient details must be provided to enable the Authority to provide the information required in the register under section 20(2)(c) of the HSNO Act.

To study genetically modified Zebrafish (Danio rerio) embryos and Caenorhabditis elegans for low-risk

research into gene function.

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Section 3: The proposed organism(s) to be developed

Section 2(1) of the HSNO Act defines what “identification” is. You must provide sufficient information to fulfill the criteria listed in the HSNO Act to enable the Authority to uniquely identify the organism in the register (as required in section 20(2)(b) of the HSNO Act).

As per sections 40(2)(a)(i)-(iv) of the HSNO Act, you must: Identify the new organism(s) (at the appropriate taxonomic level). Hint — you could start by discussing the characteristics of

the host organism and then how the proposed genetic modifications are expected to alter these characteristics.

Describe the project and the experimental procedures to be used.

Provide details of biological material to be used.

Provide details of the expression of foreign nucleic acid (if relevant).

You must describe the biological characteristics of the new organism(s). The information should be relevant to: The hazardous nature of the organism(s) that you are aware of. For example, is it a bacterium that can cause disease in

plants or humans? Will the modifications enhance the pathogenicity of a microorganism?

Which of its characteristics may enable it to escape from containment? For example, can it produce air-disseminated spores? Can it dig under fences? Can it jump or fly over high fences?

The ability of the organism(s) to form an undesirable self-sustaining population and how easy such a population could be eradicated (section 43(b) of the HSNO Act).

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Hosts

a. Danio rerio Hamilton 1822 (zebrafish) embryos less than 72 hours from

(Taxonomy: Family Cyprinidae, Order Cypriniformes, Phylum Chordata)

And

b. Caenorhabditis elegans Maupas 1900

(Taxonomy: Family Rhaditidae, Order Rhabditida, Phylum Nematoda)

Modifications

As modified by:

Standard commercially available zebrafish and C. elegans cloning and expression vectors , most of which

are non-conjugative and definitely not self transmissible.

These vectors shall only contain one or more of the following elements, and involve genetic

modifications that meet Category A experiments in the Hazardous Substances and New Organisms

(Low-Risk Genetic Modifications) Regulations 2003:

Promoters

1.1 Promoter, operator, and enhancer sequences derived from zebrafish, C.elegans, bacterial, yeast or

mammalian genes, or from bacterial or mammalian viruses.

2. Reporter Genes

2.1 Gene products that can be assayed by one or more of the following techniques:

2.1.1. Visual colour or fluorescence

2.1.2. Spectrophotometrically

2.1.3. Histochemically

2.1.4. Enzyme-linked immunosorbent assays (ELISA)

2.1.5. Thin layer chromatography

2.1.6. Liquid scintillation counting

2.1.7. Affinity purification

2.1.8. Immunological detection

2.1.9. C.elegans behavioural makers genes

3. Selectable marker genes

Well characterised genes that confer the ability to tolerate or deactivate:

3.1.1. antibiotics

3.1.2. metabolic inhibitors

Well characterised genes that confer the ability to synthesise essential metabolites and do not

produce proteins that are pathogenic or toxic to vertebrates or are involved in cellular

differentiation.

4. Origins of replication

4.1 ColE1 or the pUC origins of replication derived from Escherichia coli plasmids.

4.2 Phage f1 origin of replication.

5. Other features

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5.1. Multiple cloning site

5.2. Polyadenylation signals

5.3. Transcriptional activators

5.4. Transcriptional responsive elements

5.5. Transcriptional terminator sequences

5.6. Secretory signals

5.7. Intron sequences that function to increase gene expression

5.8. Ribosomal binding sites and/or Kozak sequences

5.9. Viral packaging signals

5.10. Viral long terminal repeat sequences

5.11. Viral genes required for replication

5.12. Cre/Lox recombinase system

With:

Genes (both sense and anti-sense constructs including nucleotide deletions and substitutions as well as

RNA interference sequences) encoding molecules involved with;

a. Diagnosis, development and modification of genetic diseases with particular reference to

neurodegenerative diseases such as Alzheimer’s, Huntington’s, Spinocerebellar ataxia and

Parkinson’s diseases

b. Metabolic pathways including fat synthesis

c. Development and growth in vertebrates (with particular reference to immunity and

haematopoiesis, gut, kidney, bone and cartilage

Such genes will typically encode:

Chaperone proteins and proteins involved in post-translational processing and protein

folding

Cell adhesion receptors, cell matrix molecules and cell membrane proteins

Signaling molecules associated with cell surface molecules (with particular reference to

proteins involved in neurodegeneration)

Structural proteins

Signal transduction molecules

Anti-Apoptotic proteins

Transcriptional factor proteins

Regulatory sequences

Transcriptional and promoter elements associated with all of the above sets of gene families

Enzymes involved in metabolism

cDNA and genomic library inserts

with:

a. Regulatory sequences associated with all of the above sets of gene families (with particular

reference to genes involved in development)

b. Genetic elements encoding protein variants with multiple amino acid repeats or those proteins

variants that may misfold

cDNA sequences encoding protein tags or fusion constructs (including fluorescent and reporter marker

proteins from Aequorea spp, and corals Discoma spp, Heteractis spp and Anthrazoa spp to determine

transgene localisation or aid protein purification

Fusion genes that would mimic and/or characterise gene translocations

Sequences encoding enzymes for assay

With the following exceptions:

Modifications will not generate a GMO that is more pathogenic, virulent or infectious to

laboratory personnel, the community or the environment

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Modifications will not generate a GMO that has a greater ability to escape from containment

than the unmodified host organism

Genes will not be derived from native biota and CITES protected species

DNA will not be directly derived from humans (unless accompanied by a specific approval

from a recognised Human Ethics Committee).

Human genes will not be derived from persons of Maori descent

Description of hosts

Danio rerio (Embryos less than 72hours)

Danio rerio embryos that are less than 72 hours old are translucent making them useful for observing

embryogenesis and development. These embryos are very vulnerable to changes in osmolarity, pH and

have an absolute requirement for water so are totally reliant on human intervention in the laboratory for

survival.

48 hours old Danio rerio embryo developing at 29.5°C inside the chorion

Currently under the HSNO (Low Risk Genetic Modification Regulations) 2003, because an embryo is a

multicellular organism, it is prescribed to be contained within a Physical Containment level 2 laboratory.

It is our assertion that when using the protocols and physical procedures documented in this application

when using this organism in the microfluidic system, a Physical containment level 1 laboratory is

sufficient to manage all risks of this organism escaping containment.

Caenorhabditis elegans

Caenorhabditis elegans is fully free-living nematode (small worm) and functions primarily as a digester of

detritus, posing no threat except to the microbes which it eats. C elegans is a non-pathogenic organism,

and working with C. elegans carries a very low level of risk.

It should be noted that dessication will severely inhibit movement of the nematode as these nematodes

rely on internal hydrostatic pressure, which acts as 'hydrostatic skeleton'. Muscle cells are tightly

connected to the external cuticle through the hypodermal cells. Contraction of muscle cells on one side

leads to bending of the rigid body. Coordinated contractions allow movement in elegant sinusoidal

Danio rerio embryogenesis at 29.5°C

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waves (hence the name C. elegans). When worms dry out they lose their internal pressure and the ability

to move (think of a balloon no longer able to maintain a rigid shape when loosing air).

C. elegans critically depends on a moist environment and has little protection against desiccation. It has

been demonstrated that if a worm strays from the agar surface of a petri plate it is found dead on the lid

or sides within ~1cm of the moist surface. Likewise, when transferring selected individual worms to a

fresh agar plate, the worm dies unless the movement is performed in less than 1 minute (personal

C.elegans experience, S. Reid). Thus any potential pathway of escape from a laboratory must involve

water, to which we have proposed multiple procedures, beyond what is already required for a Physical

Containment level 1 laboratory.

C. elegans embryos develop rapidly and hatch after 14 hours. The first larval stage is completed after

another 12 hours and the animals proceed through four molt cycles before becoming adults. Under

crowded conditions or in the absence of food larvae can choose an alternative developmental pathway

leading to the dauer larva, which does not feed but can survive adverse conditions for several months.

When life gets better normal development is resumed, the animals exit the dauer larval stage and

develop into the normal fourth larval stage before becoming adult. Adult animals are hermaphrodites

and produce both sperm and eggs or males. Over the course of 3-4 days some 300 eggs are laid. The

overall life span of C. elegans is 2-3 weeks. The short generation cycle facilitates genetic experiments and

is a major advantage for researchers working with this organism.

C. elegans life cycle at 22°C

C. elegans normally inhabits the interstices between damp soil particles or in rotting vegetation. It lives in

a film of water and is held to solid surfaces by surface tension. Locomotion is achieved by dorso-ventral

flexures of the body, which give rise to sinusoidal wave propagation along the length of the body. This

can either be in the anterior-to-posterior direction, giving rise to forward motion, or in the posterior-to-

anterior direction, giving backward motion. The head has an extra degree of freedom, in that it can make

lateral as well as dorso-ventral movements. The dorso-ventral flexures (with the consequential

sinusoidal posture of the body), combined with the surface tension forces, constrain the animals to lie on

their sides. The L1, dauer and adult stages have longitudinal lateral ridges of cuticle, the alae, which may

act to increase lateral friction and minimize sideslip.

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The thickness of the water film is quite critical; too thin or no water film results in the animals' becoming

desiccated and dying, whereas if the film is greater than their diameter they are not held down to the

surface and are unable to make any progress. C. elegans can move well on an agar surface even though

this must be quite different from its normal habitat. If there is no food available locally it will move

forward for quite long periods with occasional short intermissions of reversing. When it locates food it

starts eating and stops moving, except for short foraging excursions forwards and backwards. Eggs tend

to be laid only when the hermaphrodites have a plentiful food supply (Wallace, 1968)

The requirement for water for movement is also illustrated in the hardier dauer stage. Dessication is

NOT a signal for dauer formation (lack of food is the major trigger). Dauers can survive long periods of

no food and adverse conditions, but without water they cannot move, and hence won’t find the

conditions required to exit dauer phase and complete the life cycle of the worm.

Currently under the HSNO (Low Risk Genetic Modification Regulations) 2003, because C. elegans is a

multicellular organism, it is prescribed to be contained within a Physical Containment level 2 laboratory

It is our assertion that when using the protocols and physical procedures documented in this application

when using this organism, a dedicated Physical containment level 1 laboratory is sufficient to manage all

risks of this organism escaping containment and that use of C. elegans in a PC1 microfluidics laboratory

achieves the desired level of containment for this organism.

Description of Modifications

The range of modifications involved is bounded by specific exclusions to ensure that modifications will

not generate GMOs that have significantly different characteristics from the organisms from which they

are derived.

As such the only genetic modifications permissible are those described as Category A modifications (as

per Reg 5 (1) HSNO Low Risk Regulations, 2003) if the host was a Category A host. We note that the

Office of the Gene Technology Regulator in Australia classes low risk work with C. elegans as requiring

only PC1 containment.

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Section 4: The proposed containment system (section 40(2) of the HSNO Act)

In this section you should outline how you propose to adequately contain the new organism(s) and manage any hazards associated with the organism(s), i.e. discuss the method of containment (based on the characteristics of the organism). For example, bagging plants to prevent pollen escape or requiring spore-producing bacteria to be handled within class II biosafety cabinet. Hint—refer to the appropriate MAF/ERMA Standards and AS/NZS 2243.3:2002 (or any updated version) requirements and your facility’s containment manual where appropriate.

Are you aware of any possible adverse effects of the organism on the health and safety of the person people working the containment facility? If so, what risk mitigation strategies do you propose? For example, requiring pathogenic bacteria to be handled only by personnel using the appropriate safety gear.

If this application is for development within an outdoor containment facility: Discuss whether controls are required for inspection and monitoring before, during and after a development outdoors

within a containment facility.

Section 45A(2)(a) and (b) of the HSNO Act requires that at the completion of an outdoor development the organism and any heritable material from the organism (along with some or all of the remaining genetic elements) are removed or destroyed. Describe how you would achieve these objectives.

C. elegans is grown on moist agar plates with E. coli as a nutrient source for the nematode. All stages of

the life cycle are less than about 1mm in size, therefore microscopic viewing is required for individual

selection. C. elegans is normally transferred and manipulated individually using a flamed fine platinum

wire under a binocular scope. Worms can also be transferred in bulk when in solution using

micropipettors with disposable tips.

Zebrafish embryos and C. elegans will be loaded into microfluidic chips using micropipettors with

disposable tips within a class 2 hood

The laboratories that C elegans would be used within are MAF approved to Micro-organisms PC1

Standard at University of Auckland. We also propose further controls to limit the chance of these

organisms escaping:

Conditions set by an approval of this application is only applied to zebrafish embryos (less than 72hrs

old) used for microfluidics:

Zebrafish embryos will be loaded into microfluidics chips within a Class 2 hood and hood bench

surface wiped with ethanol at completion of work.

Once inside microfluidics chips the embryos are contained.

Transport of embryos in the between the microfluidics chips to designated work areas

(including to autoclave facilities) within a secondary closed containers to avoid the risk of

accidental spillage.

All waste water/media containing zebrafish embryos will be autoclaved or made non-viable

with Virkon or hypochlorite at least 30 min prior disposal

Following experiments, chip-based devices containing zebrafish embryos will be flushed with

100% ethanol – to avoid damaging the chips

Conditions set by an approval of this application is for all work with C. elegans:

All waste water/media containing C elegans will be autoclaved or made non-viable with 1%

(v/v) with freshly made Virkon or 5% (v/v) with freshly made sodium hypochlorite solution and

held at least 30 minutes prior to disposal

To ensure handling of C elegans in laboratories with minimal other activity and to minimise the

risk of spread, C elegans will be handled in PC1 laboratory areas dedicated to handling C elegans.

This laboratory will have access control (locks) to discourage unauthorized access. Sign on door

indicating no cleaner access.

All agar plates and disposable tips having contact with C. elegans will be autoclaved prior to

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disposal.

C. elegans will be transported between designated work areas (including to autoclave facilities)

using secondary closed containers in addition to the petri dishes (e.g. ‘click-clack’ boxes) to

avoid the risk of accidental spillage.

C. elegans will be loaded into microfluidics chips within a Class 2 hood and hood bench surface

wiped with ethanol at completion of work.

Equipment used for handling C. elegans will be autoclaved or treated with 1% (v/v) with freshly

made Virkon or 5% (v/v) with freshly made sodium hypochlorite solution and held at least 30

minutes prior to disposal

Worm picks are flamed regularly (to avoid cross contamination)

Agar plates containing C. elegans will sit in a secondary plastic container whilst on bench

surfaces or within growth incubators . Note that this is routine practice as it assists retention of a

relatively high humidity and moist agar plates necessary for C. elegans survival. Secondary

plastic container will be treated with 70% ethanol or alcohol based cleaning products after

completion of use. Bench surfaces that have been used for this work will be sprayed with 70%

ethanol or alcohol based cleaning products after completion of work

Following experiments, chip-based devices containing C.elegans will be flushed with 100%

ethanol or methanol at least 30 minutes prior to disposal in medical waste

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Section 5: Details of consultation (if applicable)

Discuss the consultation process and summarise the outcomes. Attach specific details of the consultation process (such as copies of written responses) as a separate Appendix. Discuss any adverse or beneficial effects identified during consultation in more detail in Section 6.

Rangimarie Rawiri (mandated Ngati Whatua representative on the UABSC) was contacted on 5 April

while the application was in preparation to see what interests Ngati Whatua might have in this

application.

Rangimarie indicated her preference would be to ensure the application went before the University of

Auckland Biological Safety Committee so she could take any comments from the scientific members of

the committee into consideration before formulating a response on behalf of Ngati Whatua.

The application was considered by the UABSC electronically on May 3, the members having been given

over a week to look at the application. All comments were collated and forwarded to Rangimarie Rawiri

separately. Rangimarie indicated verbally that she did not see that Ngati Whatua would have any

concerns with the application as the relevant issue involved containment level and additional specific

containment levels. A delegation of Ngati Whatua au Orakei (including Rangimarie) have visited

containment laboratories in the Containment Facility most likely to be used.

Sonia Hawea who is also a member of the Committee who advises the committee on Maori issues

indicated she could not see any issues with the application.

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Section 6: Identification of risks, costs and benefits

This section must include information on the beneficial and adverse effects, risks, costs and benefits referred to in the HSNO Act and the HSNO (Methodology) Order 1998. It is easier to regard risks and costs as being adverse (or negative) effects and benefits as beneficial (or positive) effects. You should consider both non-monetary and monetary (dollar value) costs and benefits, the distribution of their occurrence as well as who and what might be affected.

Provide a description of where the information in the application has been sourced from e.g. from in-house research, independent research, technical literature, community or other consultation. Please attach copies of all reference material cited in the application.

a) What are the nature of the adverse effects and the costs of the organism(s) that you are aware of?

i. On the environment (section 40(2)(a)(v)of the HSNO Act)

For example, could the organism adversely affect the environment while in containment? If the organism were to escape could it have an adverse effect on the environment?

Neither of the proposed organisms can survive outside aqueous environment, so containment

measures are designed to prevent release into the environment. These measures are over and above

the requirements of PC1 containment. We don’t believe that use of PC2 containment (which is either

primarily directed to containing spread of micro-organisms in aerosols or where it is directed at the

containment of terrestrial vertebrates and invertebrates) specifically addresses the containment issues

associated with water dependent small multicellular organisms.

Both of these organisms have a long record of safe laboratory use and stable genetic modification will

not alter these characteristics or alter the ability to survive outside aqueous media.

We do not believe that these organisms will survive well in the environment other than in the event

of deliberate theft of large quantities of nematodes or zebrafish embryos. Security control available

in Containment facilities (swipe card access) addresses the issue of theft.

Additional controls are directed at ensuring double contained transport and proper disposal and

cleanup.

ii. Adverse effects of occupational exposure (section 40(2)(a)(v) of the HSNO Act)

For example, could the organism adversely affect the health and safety on any person exposed in the workplace environment while in containment?

These organisms are very safe and neither will infect or colonize in animals, plants or human beings

and therefore we do not envisage any adverse effect on occupational exposure.

iii. On the relationship of Māori to the environment and the principles of the Treaty of Waitangi (section 6(d), 8 and 40(2)(b)(v)of the HSNO Act)

For example, if the organism were to escape could it have an adverse effect of potential specific importance to Māori. When identifying potential effects you should consider effects to environmental (e.g. physical impacts on native flora and fauna, water bodies, traditional food resources etc), cultural (e.g. the recognised kaitiakitanga role of Māori), health and wellbeing (e.g. specific physical and spiritual health effects), economic (e.g. the ability of Māori to develop economically) and Treaty of Waitangi (e.g. the ongoing management by Māori of their cultural or natural resources). Include any relevant issues raised or information obtained through consultation.

These organisms are to be held in containment within MAF registered containment facilities and

every effort made to ensure they do not escape. In the highly unlikely event of their escape, they

are unlikely to survive and moreover highly unlikely to have any adverse effect of potential

specific importance to Maori.

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iv. On society and the community including public health (section 40(2)(a)(v) of the HSNO Act)

For example, could the organism in containment adversely affect individuals or communities? If the organism were to escape could it have an adverse effect on society or on people’s wellbeing?

Given that these organism do not infect plants, animals and humans and given that they will be

easily contained as they have an absolute requirement for water, we believe the residual risks are

extremely low once PC1 containment and additional specific controls have been applied.

v. On the market economy (section 40(2)(a)(v) of the HSNO Act)

For example, could there be any adverse effects on the New Zealand economy at a local, regional or national level? Are there any public commercial risks or costs?

Both organisms will be used in research in containment it is difficult at this stage to see any effect on

market economy in NZ. There may be benefit (see appropriate section below) but this benefit has a

large degree of uncertainty.

vi. Are there other potential adverse effects that do not fall under sections (i) – (v)?

We do not envisage any other effects

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b) What is the nature of the potential beneficial effects associated with the organism(s) that you are aware of?

i. Beneficial effects on the environment and ecosystems

For example, could the organism beneficially affect the environment while in containment? If the organism were to escape could it have a beneficial effect on the environment?

We cannot envisage a situation where highly unlikely escape would have a positive effect on the

environment.

ii. Beneficial effects on the relationship of Māori to the environment and the principles of the Treaty of Waitangi

For example, if the organism were to escape could it have a beneficial effect of potential specific importance to Māori. As for the identification of adverse effects, you should consider effects to environmental, cultural, health and wellbeing, economic and Treaty of Waitangi. Include any relevant issues raised or information obtained through consultation.

We cannot envisage a situation where highly unlikely escape would have a positive or negative effect

on Maori

iii. Beneficial effects on public health, society and community

For example, if the organism were to escape could it have a beneficial effect on society or on people’s health and wellbeing? Could the organism in containment have benefits for individuals or communities? This might include increased knowledge.

Much of the work with zebrafish embryos and C. elegans is research aimed at understanding

vertebrate development (with particular reference to haematopoiesis, immunity, cartilage and kidney

development) and also the causes of neurodegeneration. The results of this work will be published in

peer-reviewed journals. While it is highly likely that this research will add to the sum total of

knowledge about development and neurodegeneration, it difficult to state with any certainty

whether this increase in total knowledge will have any immediate effect (i.e. development of novel

therapy).

iv. Beneficial effects on the market economy

For example, could there be any beneficial effects on the New Zealand economy at a local, regional or national level? Are there any public commercial benefits?

Other than a potential discovery which falls outside public good science funding and is able to be

patented (i.e. a novel cell sorting mechanism as a result of microfluidics research), we do not envisage

any immediate effect on the market economy. There is a very large uncertainty around whether the

research would generate a patentable discovery and whether value extracted from the resulting

intellectual property.

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v. Are there other potential beneficial effects that do not fall under sections (i) – (iv)?

We do not envisage any other beneficial effects and note that the beneficial effects noted have a level of

uncertainty as is the nature of research.

Section 7: Is there any other information relevant to the consideration of this application that has not been mentioned earlier?

We have no further information relevant to this application besides what we have already stated.

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Section 8: List of appendices, referenced material and Glossary (if applicable)

a) List of appendices attached

Appendix Number Title

1. EmbryoChip technology: An innovative mesofluidic platform for automated

manipulation of small model organisms

b) List of references used

Author Title and Journal

HR Wallace The Dynamics of Nematode Movement.

Annual Review of Phytopathology 1968

Vol. 6: 91-114

c) Glossary

Term Definition

Dauer An alternative developmental stage of nematode worms, particularly

Caenorhabditis elegans whereby the larva goes into a type of stasis and can survive

harsh conditions (lack of nutrients and overcrowding).

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Section 9: Declaration and signing the application form

In preparing this application I have: Taken into account the ethical principles and standards described in the ERMA New Zealand Ethics Framework

Protocol (http://www.ermanz.govt.nz/resources/publications/pdfs/ER-PR-05-1.pdf);

Identified any ethical considerations relevant to this application;

Ensured that this application contains an appropriate level of information about any ethical considerations identified, and provided information about how these have been anticipated or might be mitigated; and

Contacted ERMA New Zealand staff for advice if in doubt about any ethical considerations.

I have completed this application to the best of my ability and, as far as I am aware, the information I have provided in this application form is correct.

Signed

Date

Signature of applicant or person authorised to sign on behalf of applicant

Before submitting your application you must ensure that: All sections are completed.

Appendices (if any) are attached.

Copies of references (if any) are attached.

Any confidential information identified and enclosed separately.

The application is signed and dated.

An electronic copy of the final application is e-mailed to ERMA New Zealand.

http://www.ermanz.govt.nz/resources/publications/pdfs/ER-PR-05-1.pdf

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Appendix 1: EmbryoChip technology: An innovative mesofluidic platform for automated

manipulation of small model organisms

Applicant: Dr Donald Wlodkowic

Address: The BioMEMS Research Group, Department of Chemistry, University of Auckland

E-mail: [email protected]

Phone: 09 3737599 Extn 82379

Abstract:

Advances in physics, electronics and material sciences have recently led to development of miniaturized

bioanalytical systems collectively known as Lab-on-a-Chip.

Lab-on-a-Chip are the next generation of analytical laboratories that have been miniaturized to the size of a

matchbox but at the same time can automatically perform many parallel and complex biomedical

experiments faster, cheaper and much more efficiently.

In an effort to speed up the rate of drug discovery and provide a ground-breaking Lab-on-a-Chip technology

for developmental, reproductive and regenerative medicine, we aim to develop miniaturized and integrated

and fully enclosed chip-based systems for handling small model organisms. Our microfabricated technology

will automate and expand the capabilities of a wide range of biomedical research activities performed on

small model organisms offering numerous and currently inaccessible laboratory automation advantages.

Objectives:

We are developing high-throughput micro/mesofluidic technologies for automated sorting, positioning,

treatment and analysis (phenotype-based and fluorescence assays) of zebrafish embryos and larvae and other

small model organisms such as nematodes (C.elegans). We are currently developing the technology for

containing single embryos arranged in a regularly-spaced arrays on a fully-enclosed plastic chips. The chips

will be approximately the size of a standard microscope slide (about 25x75 mm) provided with means for

infusing solutions across the contained embryos and small model organisms. There will be at most

approximately 100 embryos, manipulated in circuitry of small channels and individual traps less than 1.5

mm in width and less than 50 mm in lengths. We are also working on integration of the chip-based

technologies with optoelectronic sensors and microelectronic controllers to provide fully integrated small

model organism handing system. Furthermore, we are developing an autonomous and user friendly software

and hardware interface for system laboratory automation and data acquisition on a large scale.

Concept of miniaturized bioanalysis on the

Lab-on-a-Chip devices

Microfluidic Lab-on-a-Chip device.

mailto:[email protected]

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To validate the performance of our innovative microchip technologies we will perform a range of proof-of-

concept biological experiments. This will include the most commercially viable applications such as (i)

multidimensional fluorescence imaging on living embryos (ii) supravital environmental scanning electron

microscopy (ESEM), (iii) RNAi microinjections and (iv) drug screening / toxicity assessment using

Zebrafish embryos and larvae. During experimentation biological specimens will be fully contained in chip-

based devices.

Significance and expected results:

Automated handling and sorting of single zebrafish embryos and larvae, and other small model organisms

such as Caenorhabditis elegans oocytes is still a challenging task. The cumbersome manual procedures

commonly employed in biomedical research are time consuming and error prone, limiting reproducibility

and introduction of industry standards. Moreover, the lack of technologies that combine automated

positioning, sorting, as well as pharmacological, mechanical and genetic manipulation, and analysis, of

single zebrafish embryos remains the key obstacle to high-throughput organism-based phenotypic assays for

drug discovery. Embryo and larvae handling, sorting and treatment is still preformed manually under static

microtiter plate-based conditions, limiting research productivity.

Applications of EmbryoChip Technology

We envisage that embryo sorting, capture, culture and analysis in microfluidic system, where the most tasks

are performed automatically without disturbing the embryo, and without sudden changes to embryo

environment, will prove to be better than conventional static culture. This will offer numerous advantages

currently inaccessible for pharmacology, developmental and regenerative medicine. Such integrated and

automated systems are currently unavailable.

Electronic sensors and actuators developed

for integration with microfluidic Lab-on-a-

Chip devices

Prototype of the fully enclosed microfluidic chip for

immobilization of zebrafish embryos. Chip was

fabricated using the biocompatible soft polymer PDMS

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This project will thus enable several unique capabilities over the existing technologies: (i) low cost and

portability; (ii) automated positioning of embryos at various developmental stages for cellular-resolution

imaging without the need of manual embryo handling; (iii) gentle physical immobilisation of embryos in

traps of adjustable sizes for precise focusing, laser nanosurgery and microinjections; (iv) staining or

treatment without displacing the embryos; (v) on demand single- or multiple-embryo recovery in real-time;

(vi) highly controllable fluidic microenvironment; (vii) spatial segregation of developing embryos to avoid

embryo-to-embryo interaction; (viii) applicability of customized image and data analysis software, allowing

address designation to each embryo; (ix) automation-compatible interface for both low- and high-throughput

analysis; (x) sorting of individual embryos and larvae at various developmental stages.

Materials & Methods:

Design and mathematical modelling will be performed using AutoCAD 2010, Corel Draw X3 and COMSOL

Multiphysics 3.4 software packages respectively. Modelling-guided prototyping will be performed using

high-speed CO2 laser prototyping system (Universal Laser Systems, USA) and soft-photolitography

techniques. Biocompatible polymers (PMMA and PDMS) will be utilized for multilayer 3D chip designs.

Hydrodynamic, micromechanical and vacuum assisted embryo immobilization technologies will be

developed and tested for throughput, efficiency and biocompatibility. Additional pneumatic single embryo

recovery system will be integrated for post-treatment recovery and sorting of selected embryos on 3D

multilayer devices.

New chips for sorting small model organisms and embryos will be also designed and fabricated as above but

with micromechanical and vacuum assisted modules for in-flow sorting of single embryos and larvae.

During the initial stages computer-controlled syringe pumps, peristaltic pumps and motorised time-lapse

fluorescence microscopes (Nikon Eclipse Ti-E and Leica M165FC) will be used to operate the chips and

acquire data. Subsequently, proof-of-concept analog electronic interface will be designed to increase

automation. This will include use of miniaturized CCD imaging modules (Celestron, USA), micromotor

pumps (ColeParmer, USA), on chip heaters (ColeParmer, USA) and solenoid valves (ColeParmer, USA).

Vacuum Actuated Trapping Microarray (VATM)

Multilayer 3D chip designs with integrated vacuum Actuated Trapping Microarray (VATM) technology

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