An Overview Of Transgenic Fish - Alison Van Eenennaam

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An Overview of Transgenic FishAlison Van Eenennaam, Ph.D.Cooperative Extension SpecialistAnimal Biotechnology and GenomicsDepartment of Animal ScienceUniversity of California, Davis(530) ence.ucdavis.edu/animalbiotech/ANS 131 5/24/2011Animal Biotechnology and Genomics Education

Outline What is biotechnologyIntroduction to transgenic fish – productsbeing developedConcerns about transgenic fishRegulatory structure being developedAquAdvantage salmonContainment approachesMethods for reproductive containment oftransgenic fishAnimal Biotechnology and Genomics Education

What is Biotechnology ?BiotechnologyTechnology based onbiology. The application ofscience and engineering toliving organisms.Animal Biotechnology and Genomics Education

56% of Americans oppose scientific researchinto the genetic modification of /2005summary.pdfAnimal Biotechnology and Genomics Education

Not breeding Just unnaturalgenetic modification of animals .Animal Biotechnology and Genomics Education

What is a genetically engineeredorganism ?Genetically engineered organisms(GMOs, transgenic, GE) can be definedas those which have receivedsequences of DNA by artificial means(recombinant DNA techniques),followed by integration of one or moreof the novel sequences into theirchromosomal DNAMaclean and Laight. 2000. Transgenic fish: an evaluationof the risks and benefits. Fish and Fisheries 1:146-172Animal Biotechnology and Genomics Education

Animal Biotechnology and Genomics Education

Animal Biotechnology and Genomics Education

Animal Biotechnology and Genomics Education

Animal Biotechnology and Genomics Education

Animal Biotechnology and Genomics Education

Animal Biotechnology and Genomics Education

Animal Biotechnology and Genomics Education

World Record 97 lb 4 oz. chinook salmon(Les Anderson, Kenai River, AK 1985)Animal Biotechnology and Genomics Education

Growth-enhanced Atlantic SalmonAnimal Biotechnology and Genomics Education

Same-age siblings – one carrying ahemizygous copy of the transgeneAnimal Biotechnology and Genomics Education

Current and potentialapplications of transgenic fish Growth enhancementFreeze resistance and cold toleranceSalinity toleranceDisease resistanceMetabolic modificationImproved product for the consumerSterilityFishpharming – production ofpharmacological proteinsMaclean and Laight. 2000. Fish and Fisheries 1:146-172Animal Biotechnology and Genomics Education

Transgenic Growth-Enhanced TilapiaMaclean and Laight. 2000. Fish and Fisheries 1:146-172Animal Biotechnology and Genomics Education

Transgenic Growth-Enhanced LoachHeterozygoteWild-typeNam et al. 2002. Aquaculture 209:257-270Homozygote(androgenetic)Animal Biotechnology and Genomics Education

Objections to transgenic fish The procedure used to produce the fish isunnatural and therefore undesirableTransgenes could confer undesirable as well asdesirable new properties on the fishTransgene incorporation could lead to othergenetic problems (pleiotropic effects)Novel proteins could be allergensThe fish, although not interbreeding, could beviewed as equivalent to an introduced alien speciesFish might interbreed with the wild native fish andcause ecological harmMaclean and Laight. 2000. Fish and Fisheries 1:146-172Animal Biotechnology and Genomics Education

Traits that determine net fitness Juvenile viability (chances of surviving tosexual maturity)Adult viability (chances of surviving toprocreate)Fecundity (number of eggs produced by afemale)Fertility (number of eggs successfullyfertilized by male sperm)Mating success (success at securing mates)Age at sexual maturityMuir and Howard. 2001. PNAS 96:13853-13856Animal Biotechnology and Genomics Education

Gene Flow - The Spread ScenarioPEW 2003. Future Fish: Issues in Science and Regulation of Transgenic FishAnimal Biotechnology and Genomics Education

Gene Flow - The Purge ScenarioPEW 2003. Future Fish: Issues in Science and Regulation of Transgenic FishAnimal Biotechnology and Genomics Education

Gene Flow - The Trojan gene(Muir and Howard, 1999)Occurs if thetrait bothincreasesmale matingsuccess ANDlowers theviability oftransgenicoffspring ORIncreasesjuvenileviability butreducesfertilityPEW 2003. Future Fish: Issues in Science and Regulation of Transgenic FishAnimal Biotechnology and Genomics Education

Gene Flow – Environmental concernsDepending on the interaction between thesix fitness components, the risk of gene flowcan range from none to significant Consequences vary case by case dependingon the most likely gene flow scenario and theecological characteristics of the transgenic fishand the fish community that it might affect DIFFICULT (maybe IMPOSSIBLE)TO PREDICT BEFORE THE FACT !!!! Animal Biotechnology and Genomics Education

Harvest of Fear video pieceAnimal Biotechnology and Genomics Education

First GE animal for food to market?AquAdvantage salmonAnimal Biotechnology and Genomics Education

Fish reach adult size in 16 to 18months instead of 30 monthsAnimal Biotechnology and Genomics Education

There was no regulatoryparadigm to approve GEanimals in 1993 .“In a letter to the FDA dated April 26, 1993,AquaBounty Technologies (then A/F Protein)initiated discussions with the FDA seekingregulatory guidance for development andapproval of a GE Atlantic salmon intendedto grow faster than conventionally bredAtlantic salmon (Entis, E. 2011. Personalcommunication)”Animal Biotechnology and Genomics Education

In 2009 FDA issued a finalguidance on regulations forGE animalsIn January 2009, the Food and DrugAdministration issued a final guidance forindustry on the regulation of geneticallyengineered (GE) animals (had 28,000 publiccomments on draft guidance – mostly ethical) FDA plans to regulate GE animals under thenew animal drug provisions of the Federal Food,Drug, and Cosmetic Act (FFDCA), and FDA’sregulations for new animal drugs. ry/UCM113903.pdfAnimal Biotechnology and Genomics Education

“New Animal Drug” approach“Drugs are articles intended to affectthe structure or function of the body ofman or other animals” The expression product of the newconstruct (e.g. growth hormone) is alsoconsidered to be the new animal drug Application process requires that thedeveloper demonstrate that no harmcomes to individuals who use the drugunder prescribed conditions PEW 2003. Future Fish: Issues in Science and Regulation of Transgenic FishAnimal Biotechnology and Genomics Education

Animal Biotechnology and Genomics Education

FDA public Veterinary Medicine Advisory Committee(VMAC) Meeting was held September 19-20th, 2010Labeling meeting was held September 21st, 2010Animal Biotechnology and Genomics Education

Elliot Entis, Founder of AquaBounty at the PublicHearing on the Labeling of Food Made fromAquAdvantage Salmon, September 21st, 2010Animal Biotechnology and Genomics Education

Product Definition for theAquAdvantage SalmonProduct IdentityTriploid hemizygous, all-female Atlantic salmon (Salmo salar) bearinga single copy of the α-form of the opAFP-GHc2 rDNA construct at theα-locus in the EO-1α lineage.ClaimSignificantly more of these Atlantic salmon grow to at least 100 gwithin 2700 deg C days than their comparators.Limitations for UseThese Atlantic salmon are produced as eyed-eggs in Canada forgrow-out only in the FDA-approved physically-contained fresh waterculture facility located in Panama.Animal Biotechnology and Genomics Education

Food/Feed Safety: Does food or feed from the GEanimal pose any risk to humans or animalsconsuming edible products from GE animalscompared with the appropriate non-transgeniccomparators?Conclusion of food/feed safely evaluations:“We therefore conclude the food fromAquAdvantage Salmon (the triploid ABTsalmon) that is the subject of this applicationis as safe as food from conventional Atlanticsalmon, and that there is a reasonablycertainty of no harm from the consumption offood from this animal. No animal feedconsumption concerns were identified”.Page 62, AquAdvantage Briefing packet. yCommittee/UCM224762.pdfAnimal Biotechnology and Genomics Education

Environmental Safety: What is thelikelihood that AquAdvantage Salmon willescape the conditions of confinement?Where will the AquAdvantage Salmon be raised?If approved, the AquAdvantage Salmon will be raised ininland tanks. They will not be raised in ocean net pens.Any change would require a new application and approval.There are multiple and redundant physical and mechanical barriers in placein the water systems at the PEI egg production and Panama grow-outfacilities to prevent the accidental release of eggs and/or fish to nearbyaquatic environments. These barriers have been designed specifically toprevent the escape of different life stages of AquAdvantage Salmon. Bothfacilities have a minimum of three to five mechanical barriers in place for allinternal flow streams which release water to the environment. Standardsand has been verified by an FDA inspection or site visit. Therefore, thelikelihood is considered very low that AquAdvantage Salmonwill escape from confinement at these sites.Animal Biotechnology and Genomics Education

Animal Biotechnology and Genomics Education

Letter asking to“halt allproceedings”related to GEsalmon signedby 11 Senators,and a similarone signed by29 members ofCongress sentto FDACommissionerHamburg9/28/10Animal Biotechnology and Genomics Education

This letterdated 2/1/11was signed by Friends of theEarth, Earthjustice, Greenpeace, Oceana, OceanConservancy, PewEnvironmentalGroup, Union ofConcernScientistsAnimal Biotechnology and Genomics Education

2/2/11 bill toban GEsalmon wasintroducedby:Senator MarkBegich, D-AKSenator LisaMurowski, on/Animal Biotechnology and Genomics Education

Retrieved from “AquAdvantage” image search on webGGG 293 5/16/2011Animal Biotechnology and Genomics Education

Retrieved from “AquAdvantage” image search on webFrankenfishAnimal Biotechnology and Genomics Education

1. Government regulators should include ethical and moralconsiderations, in addition to scientific evaluation of risks andbenefits, when making regulatory decisions about cloning orgenetically modifying animals.2. Though ethical and moral considerations are important,government regulators should consider only scientificevaluation of risks and benefits when making regulatorydecisions about cloning and genetically modifying animals.1. Morals / Ethicsand Science53%10%63%2. Science only18%9%27%10%Not Sure10%Feel that way e/2005summary.pdfAnimalAnimal BiotechnologyGenomics and andBiotechnologyGenomics EducationEducation

What about California ?Animal Biotechnology and Genomics Education

Existing regulations governinggenetically-engineered aquaticorganisms in CACA Senate Bill 245 “In the waters of the PacificOcean that are regulated by this state, it isunlawful to spawn, incubate, or cultivate anyspecies of finfish belonging to the familySalmonidae, transgenic fish species, orany exotic species of finfish.”[Approved by Governor October 12, 2003. Filed with Secretary of StateOctober 12, 2003.]Animal Biotechnology and Genomics Education

Existing regulations governinggenetically-engineered aquaticorganisms in CAAdditionally, California Fish and Game departmentregulations require the possession of a permit toraise GE fish in contained onshore systems inCalifornia. This regulation applies to all transgenicfish based on the assumption that they “pose athreat to native wildlife, the agricultural interestsof the state or to public health.”February 2003Animal Biotechnology and Genomics Education

To date several permits have been grantedto medical & scientific research laboratories(9) Transgenic Aquatic Animals. The department may issue permits for importation, possession, transportation orrearing of, or research on, transgenic aquatic animals pursuant to the following terms and conditions:(A) All transgenic aquatic animals shall be held, raised, and transported in a closed-water system or in a systemwhich treats effluent discharge from the facility with a disinfection system adequate to ensure against theinadvertent release of live animals. A closed-water system means that there is no discharge to waters of the state.Municipal treated sewage systems are not considered waters of the state. The Commission may grant anexception to this regulation if it is determined that doing so shall not pose a significant risk to the waters or wildlifeof the state.(B) Access to facilities containing transgenic aquatic animals must be restricted through means determined to beadequate by the Department to assure against unauthorized removal of animals.(C) Movement of live transgenic aquatic animals from facilities is prohibited unless specifically permitted by theDepartment.(D) Release of transgenic aquatic animals or their progeny into waters of the state is prohibited.(E) If transgenic aquatic animals are held with non-transgenic animals of the same species, all such animals thatcommingle with transgenic animals shall be treated as transgenic for the purposes of regulation and may not beintroduced into waters of the state. Nontransgenic individuals that can be individually identified as nontransgenicmay be exempt from this provision with prior department approval.(F) In addition to any other penalty provided by law, any unauthorized release of transgenic aquatic animals or theirprogeny into the waters of the state may be subject to the penalties provided for under Fish and Game CodeSections 2125, 12007, and/or 12023(G) A university, college, governmental research agency or other bona fide scientific institution, as determined bythe department, may apply for an expedited permit review under 671(b)6 of this section by demonstrating that theymeet or exceed the requirements stipulated in subsections (A) through (F) as part of a federal program or permit,for example, National Institute of Health guidelines administered by an Institutional Animal Care and UseCommittee (IACUC). Such institutions shall have 120 days from the date of adoption of these regulations to submitsupporting documentation for an initial permit.Animal Biotechnology and Genomics Education

GloFish Animal Biotechnology and Genomics Education

FDA statement on Glofish“Because tropical aquarium fish are not used forfood purposes, they pose no threat to the foodsupply. There is no evidence that thesegenetically engineered zebra danio fish pose anymore threat to the environment than theirunmodified counterparts which have long beenwidely sold in the United States. In the absenceof a clear risk to the public health, the FDA findsno reason to regulate these particular fish.”December 9, 2003Animal Biotechnology and Genomics Education

GloFish – barred on ethicalgrounds in California - “just a pet”Animal Biotechnology and Genomics Education

“just a floral arrangement”Animal Biotechnology and Genomics Education

Animal Biotechnology and Genomics Education

The “Precautionary” principleAlthough there is no consensus definition of what istermed the precautionary principle, one oft-mentionedstatement sums it up: "When an activity raisesthreats of harm to human health or theenvironment, precautionary measuresshould be taken even if some causeand effect relationships are not fullyestablished scientifically."In other words, actions taken to protect the environmentand human health take precedence. Therefore, someadvocates say, governments should immediately ban theplanting of genetically modified crops, even thoughscience can't yet say definitively whether they are adanger to the environment or to consumers.Animal Biotechnology and Genomics Education

Possible future advantages What if biotechnology could be used to reducethe amount of mercury or other toxiccompounds that fish accumulate from theirenvironment ?What if you could remove the allergens thatmake shellfish intolerable for some consumers ?What if you could use transgenic fish to detectenvironmental contaminants ?“BE CAREFUL NOT TO OBSCURE THEBENEFITS NOT READILY APPARENT INTHE FIRST GENERATION OF PRODUCTS”PEW 2003. Future Fish: Issues in Science and Regulation of Transgenic FishAnimal Biotechnology and Genomics Education

Who gets to decide ?Animal Biotechnology and Genomics Education

Need for biological or geneticcontainment of GE fishIt is becoming clear that unless totalphysical containment is available, thebenefits of transgenic manipulation infish will be dependent upon thedevelopment of methods toreproductively isolate transgenic fishAnimal Biotechnology and Genomics Education

What can science do ?Approach taken by Aqua Bountyhas been to produce monosex(female), triploid linesAnimal Biotechnology and Genomics Education

Induced triploidy ? Effectiveness varies greatly (10-95%)Difficult to screen every fish individuallyand cannot check eggsRelease of triploid fish into theenvironment presents certain hazards asthey still have enough hormones to enterinto normal courtship and courtingbehavior and so could interfere withreproduction of wild fishSterile fish survive longer so could increasepopulation pressure on wild fisheriesAnimal Biotechnology and Genomics Education

DEVELOPMENT OF METHODS FOR THEBIOLOGICAL CONTAINMENT OFTRANSGENIC FISHAnimal Biotechnology and Genomics Education

Animal Biotechnology and Genomics Education

Use genetic engineering as a method to remove thetransgene from the germ cells, but leave itexpressing in the somatic cells.F0 loxP-ziwi:eGFP 22 dpfAnimal Biotechnology and Genomics Education

Mosaic expression in founderfishF0 female line construct 48 hpfAnimal Biotechnology and Genomics Education

Uniform expression in F1 fishF1 Female line construct (hemizygous) 48 hpfAnimal Biotechnology and Genomics Education

Mosaic expression in FLPe RNAinjected fishWong, A.C., B. W. Draper, and A. L. Van Eenennaam. 2011. FLPe functions in zebrafish embryos. Transgenic Research. 20:409-415.Animal Biotechnology and Genomics Education

Final thoughts . The environmentally-responsible utilization oftransgenic techniques in aquaculture willrequire the development of methods forbiological containment.The development of containment techniqueswill help to ameliorate some of the gene flowconcerns about the interbreeding ofgenetically engineered fish with nativepopulations.Animal Biotechnology and Genomics Education

Conclusions No genetically engineered animal species haveyet been commercializedEven if approved at the Federal level, there areadditional State-specific regulations related tothe rearing and possession of geneticallyengineered fish in CaliforniaUnlikely there will be any GE aquaculture oranimal agriculture in CA in foreseeable futureAnimal Biotechnology and Genomics Education

Animal Biotechnology and Genomics Education

Animal Biotechnology and Genomics Education An Overview of Transgenic Fish Alison Van Eenennaam, Ph.D. Cooperative Extension Specialist Animal Biotechnology and Genomics Department of Animal Science University of California, Davis (530) 752-7942 . food from this animal. No animal feed consumption concerns were identified”. .

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