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

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 ER-AF-N03-4 09/09

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 Genetic modification of animal and lines for research and teaching purposes

b) Organisation name

University of Otago

c) Postal Address Department of Microbiology and Immunology, University of Otago P O Box 56 Dunedin

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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).

The University of Otago is a research-intensive university. This application seeks approval to genetically modify animal and human cell lines to provide researchers and students with tools to explore the role and function of genes. These develpoments will underpin research into distinct areas of but are unified by their use of standard laboratory methodology, including the use and replication-deficient viral vectors, to transfer nucleic acids into animal or human cell lines. The ability to genetically modify these cell lines in this way will help researchers to determine gene function, and more specifically how individual genes and groups of genes influence biological processes in vivo. This research is designed to generate and apply new knowledge of processes involved in: 1. Cell growth, metabolism, differentiation and development; 2. Biological responses to environmental and chemical stress; 3. Host-pathogen and host-commensal interactions; and 4. The causation of disease The animal and human cell lines to be modified will be established cell lines obtained from commercial sources or from reputable scientific laboratories, or will be primary cell lines developed with the approval of an appropriate Ethics Committee. The cell lines may include embryonic and induced pluripotent stem cell lines of animal species and induced pluripotent stem cell lines derived from , but will not include lines derived from humans. In many cases the genetic manipulation will involve a complete gene sequence from one species being introduced into a cell line derived from the same or a different species to enable changes in the biology or of the recipient cell line to be correlated with the function of the transferred gene. In some cases DNA sequences will be introduced into a cell line to investigate function by silencing endogenous genes within that host. A further scientific goal of this research involves expression of genes by modified cell lines to allow purification and biochemical characterisation of recombinant , or to produce replication-deficient viral vectors. In other cases, “reporter” genes will be introduced into a cell line in order to allow the expression of a gene within it to be monitored by various techniques (e.g. fluorescent microscopy for cell lines expressing green fluorescent ) that detect expression of the . The broad nature of these studies requires approval to modify a wide range of animal and

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human cell lines with a variety of genes from different organisms. However in all cases the modifications will be restricted by virtue of the fact that genes transferred to any cell line will not lead to the production of infectious particles such as prions or replication-competent and will not increase the risk of the cell lines to humans. The cell lines are completely reliant on human intervention for survival and would not survive outside of controlled conditions in the laboratory. Therefore, the nature of containment is such that the risk of escape and subsequent infection is negligible. The benefits from the development of these cell lines will be to add to the body of scientific knowledge of processes involved in growth, metabolism, differentiation, disease and response to stress, and knowledge that may lead to the development of new treatments or diagnostic tools for various diseases of humans and animals. 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 develop genetically modified cultured animal and human cell lines for research and teaching purposes to generate knowledge of processes involved in growth, metabolism, differentiation, development, stress response and disease.

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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 (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).

Organisms A. Animal cell lines derived from the species listed in Appendix 1. B. Human cell lines. The animal and human cell lines to be modified will be established cell lines obtained from commercial sources or from reputable scientific laboratories, or will be primary cell lines. Primary cell lines will be developed with the approval of an appropriate Ethics Committee and following appropriate Maori consultation or will be obtained from commercial sources or from reputable overseas scientific laboratories that developed the lines under ethical approval. The cell lines to be developed may include stem cell lines, including embryonic stem cell lines and induced pluripotent stem cells of animal species and induced pluripotent stem cells derived from humans, but will not include human embryonic stem cell lines. Any use of the genetically- modified stem cell lines to develop genetically-modified animals is not covered in this application and will only be done under an appropriate HSNO Act approval that covers the development of genetically-modified animals. The term "cell lines" refers to cultured cells derived from multicellular . Cell lines can be derived from tissues for ex vivo culture in several ways, but in all cases their establishment will be under fastidious controlled conditions. Primary cell lines are cell lines that are cultured directly from an organism. With the exception of some derived from tumours, most primary cell lines have limited lifespan. After a certain number of doublings, the cells stop dividing though they may retain viability. An established or immortalised cell line has acquired the ability to proliferate indefinitely either through random or deliberate modification. There are numerous well-established immortalised cell lines representative of particular cell types.

Cell lines are grown in an incubator and maintained at an appropriate temperature and atmosphere particular to the cell requirements. Culture conditions may vary for each cell type, and variation of conditions for a particular cell type can result in different phenotypes being expressed.

Cell lines are considered low risk and easily containable at Physical Containment level 1 (PC1), because they require sophisticated culture systems to maintain viability. Cell lines cannot survive outside of a highly specific nutrient environment. Cell lines cannot be transmitted to other organisms without human intervention. Cell lines are relatively slow-growing, and must be maintained under sterile conditions. Modification of cell lines is highly unlikely to enhance Page 6 of 19 the survival characteristics of individual cells. Therefore the risk of cell lines escaping from containment is negligible.

This application specifically excludes cell lines known to shed viruses, virions or prions. Many of the cell lines that are the subject of this application are derived from cell lines that have been grown for many years and are very well characterised. The genetically modified cell lines developed from them are therefore highly unlikely to be carrying unidentified inseparable organisms.

Modifications

All developments in containment using the cell lines can be safely and adequately contained under PC1 or PC2 containment, as described in the University of Otago Containment Manual.

Developments will involve the creation of both stably transfected cell lines and transiently transfected cell lines. Transient does not require recombination (whereby the DNA becomes inserted into the genome of the host cell), and is not intended to produce cells capable of reproducing the introduced DNA. The introduced DNA is short-lived, and cells are not maintained for long periods of time.

Modifications to cell lines under a minimum of PC1 containment will only include: (a) nucleic acid that is characterised to the extent that— (i) its sequence is known; or (ii) its gene function is understood; and (b) the modification will not— (i) result in the production of replication-deficient viral vectors.

Modifications to cell lines under a minimum of PC2 containment will include modifications that result in the production of replication deficient viral vectors. Modifications to cell lines under PC1 or PC2 containment will not: (i) result in a genetically modified organism that is more pathogenic, virulent, or infectious to laboratory personnel, the community, or the environment; or (ii) result in the genetically modified organism having a greater ability to escape from containment than the unmodified host organism; or (iii) result in a genetically modified organsim that produces infectious particles such as prions, virions or replication-competent viruses.

Replication-deficient viral vectors that may be generated under PC2 containment in this research may be derived from , Parvoviruses, Adenoviruses and Papillomavirus and are classified here as vectors and not organisms due to their inability to replicate.

Types and sources of additional genetic material: Reporter gene inserts expression in cell lines may be monitored using reporter genes in place of, or in addition to, candidate gene inserts.

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Candidate gene inserts DNA for the candidate genes and mutants thereof will be sourced commercially or from reputable scientific research laboratories or cloned from genomic DNA (with or without prior amplification by PCR), or from DNA reverse-transcribed from RNA and then amplified by PCR. The genes of interest will comprise of viral, eukaryotic and prokaryotic genes and mutants thereof (e.g. deletion, substitution and chimaeric mutant genes).

Additional vector elements Expression constructs might also include: eukaryotic, prokaryotic and viral enhancers/promoters silencing elements (short interfering RNA, short hairpin RNA recombination sites internal ribosomal entry sites (IRES) sequences for fusion protein tags polyadenylation signals genes for antibiotic resistance other regulatory elements that are components of existing or new commercially available vectors

Genetic material will not be from primary human sources in New Zealand unless appropriate ethical and Maori consultation has occurred. Consultation with Maori has been initially been carried out for use of genetic material from native flora and fauna, and will continually be updated and consulted when native flora and fauna donor DNA is to be used.

Genetic material sourced from CITES-listed organisms shall not be used without written approval from the Department of Conservation.

Further restrictions to the modifications to the cell lines include:

developments involving the expression of genes encoding known or suspected vertebrate with an LD50 < 100 µg/kg: developments involving viral vectors whose host range includes human cells and that contain one or more inserted nucleic acid sequences coding for a product that can lead to uncontrolled mammalian cellular proliferation or be toxic to mammalian cells, or both: developments involving or resulting in viral genomes, viroids, or fragments of a genome capable, in the host/vector system used, of giving rise to particles naturally infectious and normally able to cause disease in humans, animals, plants, or fungi developments involving recombinations between whole viral genomes, viroids, or complementary fragments of these genomes, where one or more fragments contain one or more virulence determinants or pathogenic determinants, including developments that can alter the host range of a pathogen or that increase the virulence or infectivity of the .

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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 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.

The experiments described in this application will be carried out in laboratories in an approved containment facility in accordance with the ERMA-approved University of Otago Containment Manual. These laboratories comprise PC1 and PC2 containment areas, which meet regulatory requirements and are inspected regularly.

(i) PC1 laboratory Research involving animal and human derived cell lines will be carried out under a minimum of PC1 containment as described in the University of Otago Containment Manual. Access is restricted to appropriately-trained staff and students.

(ii) PC2 laboratory Research involving animal or human cell lines to produce replication deficient viral vectors will be carried out in a minimum of PC2 containment in accordance with that described in the University of Otago Containment Manual. Access is restricted to appropriately-trained staff and students.

Application of genetically modified cell lines to vertebrate animals will be with the approval of an appropriate Animal Ethics Committee and will under a minimum of PC2 containment in an approved Containment Facility for Vertebrates, in accordance with the University of Otago Containment Manual. Genetically-modified cell lines will not be applied to humans.

When no longer required for research or teaching purposes, the genetically modified cell lines will either be destroyed or placed into frozen storage.

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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.

The Ngai Tahu representative and the Lay representative on the University of Otago IBSC were consulted on this application. Both made comments to improve the clarity of the application and these were incorporated into the application as submitted. Both supported the submission of 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? Potential adverse effects on the environment are only possible if a cell line escapes containment. All cell lines derived from animals or humans are completely reliant on human intervention for survival and the modifications will not enhance the pathogenicity, virulence or ability of the organisms to survive outside of containment. Therefore, the nature of containment is such that the risk of escape and subsequent infection is negligible.

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? The risks of adverse effects on staff are minimal because staff working with these organisms in PC1 and/or PC2 laboratories are trained on the risks involved in working with cell lines, including any risks for individuals who are immunosuppressed or immunocompromised.

All „open container‟ manipulations of replication deficient viral vectors will be performed in a Biological Safety Cabinet in accordance with the University of Otago Containment Manual.

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.

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The nature of cell lines and their containment is such that the risk of escape is negligible and as such no adverse effects of specific importance to Māori have been identified.

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? Establishment of a self-sustaining population is determined to be almost impossible; cell lines are critically dependant on nutrients and controlled environments supplied by laboratories. Modifications will not enhance the pathogenicity, virulence or ability of the organisms to survive outside of containment. Therefore, the nature of containment is such that the risk of escape and subsequent infection are negligible.

In the unlikely event of a possible breach of containment, the University of Otago Containment Manual has a contingency plan. This includes notifying the MAF Supervisor of the containment facility as soon as possible and at least within 24 hours of noticing a breach of containment. Procedures to manage spills of any cell lines within and outside the facility and for fire, personnel decontamination, theft and sabotage are also addressed in the containment manual.

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? No potential adverse effects were identified as this research is conducted in containment under strict guidelines and this research involves laboratory-adapted cell lines.

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

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No other adverse effects were identified.

Page 13 of 19 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? As the cell lines would not survive if released into the environment, no beneficial effects on the environment or ecosystems are envisioned.

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.

No potential beneficial effects if the organisms were to escape have been identified in this area.

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. If the organisms were to escape, there would be no beneficial effect on society or on people‟s health or wellbeing. However, research undertaken using the modified cell lines is likely to generate knowledge that will have a beneficial effect on society or on people‟s health or wellbeing. The development of the organisms for teaching purposes will benefit society through contributing to the development of a society informed about genetic sciences.

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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?

As this is an application for research in containment no potential beneficial effects in this area have been identified.

v. Are there other potential beneficial effects that do not fall under sections (i) – (iv)? Other potential beneficial effects identified are an increase in the body of scientific knowledge associated with processes involved in: 1. Cell growth, metabolism, differentiation and development; 2. Biological responses to environmental and chemical stress; 3. Host-pathogen and host-commensal interactions; and 4. The causation of disease

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

This application is designed to replace all the low-risk approvals for animal and human cell lines that have previously been approved by the IBSC at the University of Otago.

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

a) List of appendices attached

Appendix Number Title

b) List of references used

Author Title and Journal

c) Glossary

Term Definition

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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.

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Appendix 1: Organisms that cell lines may be derived from

Source Organism Common name Taxonomic family Human cell lines: Homo sapiens (Linnaeus, Human Hominidae 1758)

Insect cell lines Spodoptera frugiperda Fall armyworm Noctuidae (Smith & Abbot 1797) Drosophila melanogaster fruit fly Drosophilidae (Meigen, 1830) Trichoplusia ni (Hubner cabbage looper Noctuidae 1802) Bombyx mori silkworm Bombycidae Aedes aegypti mosquito Culicinae Apis mellifera Honey bee Apidae Bombus sp. Bumble bee Apidae Acyrthosiphon pisum (Harris Pea aphid Aphididae M, 1776)

Mammalian cell lines Mus musculus (Linnaeus, House mouse Muridae, 1758) Mus spretus (Lataste, 1883) Algerian mouse Muridae

Rattus rattus (Linnaeus, black rat Muridae 1758) Rattus norvegicus brown rat Muridae (Berkenhout, 1759) aethiops African green monkey Cercopithecidae (Linnaeus, 1758) Ovis aries (Linnaeus, 1758) sheep Bovidae Bos taurus (Linnaeus, 1758) cattle Bovidae Canis familiaris (Linnaeus, dog Canidae 1758) Oryctolagus cuniculus European rabbit Leporidae (Linnaeus, 1758) Sylvilagus sp (Gray, 1867) cottontail rabbit Leporidae Cricetulus griseus (Milne- Chinese hamster Cricetidae Edwards, 1867) Cricetus cricetus (Linnaeus, black-bellied hamster Cricetidae 1758) Cavia porcellus (Linnaeus, guinea pig Caviidae 1758) Felis catus Domestic cat Felinae Cervus elaphus Red deer Cervidae Pan troglodytes chimpanzee Hominidae Mustela lutreola European mink Mustelinae Mustela vison American mink Mustelinae Sus scrofa pig Suiformes Mesocricetus auratus Syrian golden hamster Cricetidae Page 18 of 19

Amphibian cell lines Xenopus laevis Clawed frog Pipidae

Fish cell lines Danio rerio zebrafish Cyprinidae Oncorhynchus sp. Salmon Salmonidae

Avian cell lines Gallus gallus Chicken Phasianidae

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