 |
| About Bioline | All Journals | Testimonials | Membership | News |
|
||||||
|
||||||
GENERATION, CHARACTERIZATION AND RISK ASSESSMENT OF TRANSGENIC TILAPIA WITH ACCELERATED GROWTH Jose de la Fuente,^1 Oscar Hernandez,^2 Rebeca Martinez,^1 Isabel Guillen,^1 Mario Pablo Estrada^1 and Ricardo Lleonart^1
^1 Mammalian Cell Genetics Division. Center for Genetic
Engineering and Biotechnology, P.O. Box 6162. Havana 6,
Cuba.
ABSTRACT Marine Biotechnology is a fast developing area in modern Biotechnology. Recent advances in gene transfer has offered the possibility to manipulate growth in fish through the transfer of chimeric growth hormone (GH) genes. We have generated several transgenic tilapia lines by the transfer of constructs containing the tilapia GH cDNA or chromosomal gene. A transgenic tilapia line with accelerated growth was obtained and characterized. The risks associated with the work in transgenic fish and shellfish were evaluated and measures were taken to conduct new experiments before introducing transgenic tilapia into the national aquaculture program. Key words: transgenic, fish, tilapia, hormone RESUMEN La biotecnologia marina es un area de rapido desarrollo dentro de la Biotecnologia moderna. Los avances recientes en las tecnicas de transferencia de genes han posibilitado la manipulacion del crecimiento en peces mediante la transferencia de genes de hormona de crecimiento (HC) quimericos. Nosotros hemos generado varias lineas de tilapia transgenicas mediante la transferencia de construcciones que contienen el ADNc o el gen cromosomal que codifica para la HC de tilapia. Se obtuvo y caracterizo una linea de tilapia transgenica con mayor velocidad de crecimiento. Los riesgos asociados con el trabajo con peces transgenicos fueron evaluados y se tomaron medidas para realizar nuevos experimentos antes de introducir las tilapias transgenicas en los planes de desarrollo acuicola del pais. Palabras claves: transgenesis, peces, tilapia, hormona
Introduction Recent Advances in Marine Biotechnology
The main topics of interest in the field of marine biotechnology could be focused in the following areas (1, 2):
b. Metamorphosis and reproductive activity in marine organisms. c. Disease related problems in aquaculture. d. Molecular Biology, genetics and cell culture. e. Bioactive substances from marine organisms. f. Utilization of marine photosynthetic organisms for food and feed production. g. Marine based industrial materials. h. Hydrothermal vent microbiology. i. Bioluminescence.
a. Transgenic fish principles and applications
The generation of transgenic fish with chimeric growth hormone (GH) transgenes for manipulating growth performance in economically important species is the main goal of this area. The present state of the art is mainly focused on:
- The use of "all fish" transgenes. - The expression of transgenes introduced by microinjection or electroporation in fish embryos. - The study of new methods for introducing foreign DNA into fish embryos (sperm cells as vectors). - The inheritance and expression of heterologous genes. - The use of GH genes in transgenic fish experiments. - The construction of fish "bio-reactors".
This field is focused on the characterization of signal molecules, chemosensory receptors and signal transducers controlling settlement and metamorphosis of the larvae of mollusks, corals, shrimps, lobsters and other organisms. It also studies the peptide pheromones and hormones that regulate reproductive activity in various marine species. Protein fractions in the digestive tract of the mollusk Pecten maximus and in the hepatopancreas of the lobster Panulirus argus have been found to immunoreact with an antisera directed against human insulin and hGH (3, 4). Furthermore, the immunoreactive protein fractions promote growth acceleration in the larvae of the shrimp Penaeus schmitti when supplied in the diet (3) and a recent publication reports on the effect of dietary rGHs on Penaeus vannamei and other shellfish larval development. These preliminary results, together with the identification of peptide GH in mollusks, allow for the cloning and utilization of these GHs and/or fish-derived GHs (5-10) and insulin-like genes for the production of recombinant proteins for diet supplementation or for the generation of transgenic organisms. Transgenic principles for these species are to be established. However, recently we reported the transient transformation of the shrimp P. schmitti (11) as a first step for the generation of transgenic shrimps. c. Disease related problems in aquaculture
One of the major factors that will determine whether the aquaculture industry will succeed is disease control. To meet this need, many different biotechnological approaches are currently being applied in the control of diseases in fish and shellfish. They include the use of Mab and nucleic acid probes for improved diagnosis of disease and the cloning of important genes which may be subsequently expressed for subunit vaccine production or for the development of transgenic animals which are resistant to pathogens. d. Molecular Biology, genetics and cell culture
All the projects listed above have experiments dealing with Molecular Biology, genetics and cell culture. We have tested the constructs employed for transgenesis in fish cell lines and embryos to determine the appropriate regulatory sequences for transgene expression (12-15). Genetics in economically important fish species is being addressed by many groups throughout the world. In tilapia, groups in Cuba have reported good results in response to selection (16). f. Growth Hormone Gene Transfer in Fish The era of effective gene transfer in animals began in 1980 with the pioneer work of J. Gordon (17). Since then, gene transfer technology has produced a great impact in modern molecular biology and biotechnology (18, 19). In the past few years, significant progress has been made in transgenic fish studies (20, 21). In particular, many efforts have been directed towards growth manipulation in fish because of the possible impact of these improved strains in aquaculture. Growth hormone gene transfer has been accomplished in several fish species (17, 21, 22). Recently, size increases in transgenic fish have been reported in some species (Table 1). However, because of the high variability in size and weight of fish (17), results from experiments assessing growth should be cautiously taken, unless evidence correlating the expression of the GH transgene and the phenotypic effect observed are supplied. Finally, the use of fish GH genes will probably give better results than mammalian genes and cDNAs.
--------------------------------------------------------------
Table 1. Results showing growth enhancement in fish in GH
gene transfer experiments.
--------------------------------------------------------------
Gene Fish species Size Inheritance Reference
construct increase
--------------------------------------------------------------
mMT-hGH loach, carp 3-4 fold yes 23-25
RSV-tGH carp 20 % yes 26
-2% - 59% yes 27
AFP-sGH atlantic salmon 4-6 fold ND 28
RSV-bGH northen pike yes ND 29
ch-beta-
actin-hGH medaka yes yes 30
OnMT-GH1 coho salmon 11 fold ND 31
CMV-tiGH tilapia 80 % yes 32, 33
Abbreviations: mMT, mouse metalothionein promoter; OnMT,
sockeye salmon MT promoter; RSV, Rous sarcoma virus LTR; hGH,
human GH; tGH, trout GH; AFP, antifreeze protein promoter;
sGH, salmon GH; bGH, bovine GH; GH1, sockeye salmon type-1 GH
gene; chbetaactin, chicken beta-actin promoter; CMV,
cytomegalovirus; tiGH, tilapia GH.
--------------------------------------------------------------
Gene Transfer in Tilapia Oreochromis Sp. Tilapia are economically important species accounting for over 67 % of the fresh water fish production in Cuba. Gene transfer in tilapia has been reported by different authors (32, 34-37) (Table 2). However, size increase in transgenic tilapia was not reported in these species until recently, when we presented our data at the International Marine Biotechnology Conference (1994) (32).
--------------------------------------------------------------
Table 2. Gene transfer experiments in tilapia.
--------------------------------------------------------------
Gene DNA Integ- Expres- Inheri- Ref-
construct ration sion tance erence
--------------------------------------------------------------
mMT-hGH Linear, 4 kb yes ND ND 34
avianLTR-bGH Linear, 3.5 yes ND ND 36
and 8.5 kb
mMT-rGH Linear, 6.6 kb yes ND ND 35
CMV-tiGH Linear, 3.2 kb yes yes yes 32
CMV+/-INT- Linear, 6.8 yes yes yes 32
tiGH and 6.2 kb
RSV+INT-tiGH Linear, 6.5 kb yes yes yes 32
RSV-chrtiGH Linear, 7.0 kb ND ND ND Martinez,
et al. unpublished.
Abbreviations rGH, rat GH; INT, first intron from trout GH;
chrtiGH, chromosomal tiGH gene; avianLTR, LTR from an avian
retrovirus; +/-, in the precense and absence of the intron.
Other abbreviations, as in table 1.
--------------------------------------------------------------
Generation of Transgenic Tilapia with tiGH Transgenes
The possibility of accelerating growth in tilapia was assayed by the exogenous administration of recombinant tiGH. The tiGH cDNA was cloned from hybrid tilapia pituitary glands and expressed in E. coli (38) and in the Pichia pastoris yeast (Lleonart et al. unpublished). The growth of juvenile tilapia (Oreochromis sp.) was accelerated (1.4 fold in length and 1.7 fold in body weight after 21 days) when the yeast-derived r-tiGH was administrated by three intraperitoneal injections at intervals of seven days. The control group received BSA injections. For gene transfer experiments, chimeric constructs were prepared containing the tilapia growth hormone cDNA (tiGH) or chromosomal gene (chr-tiGH), 5' regulatory sequences derived from the human cytomegalovirus (CMV) or Rous sarcoma virus (RSV), polyadenylation sites from the SV40 and the first intron from the trout growth hormone gene (INT) (12, 13, 37). Employing these constructs, tiGH transient expression was obtained in mammalian and/or fish cells. In previous studies we found that in Xiphophorus A2 cells CMV enhancer-promoter was more efficient than RSV sequences and that the presence of an intron is relevant for the RSV, but not for the CMV regulatory region (15). In contrast, in zebrafish embryos, the RSV regulatory region is 17 times stronger than the CMV, and again the presence of the intron is important only for the activity of the RSV enhancer- promoter (15). These results support the hypothesis that different regulatory requirements exist in cells and embryos and suggest that chimeric constructs designed for transgenic experiments should be assayed in transient expression experiments in fish embryos. To evaluate the activity of different regulatory elements in transgenic fish, five lines of transgenic tilapia were generated (CMV>+/-INT>tiGH>SV40, CMV>tiGH>CAT>SV40 (CMV-tiGH), RSV>+INT> tiGH>SV40, and RSV>chr-tiGH) by direct microinjection of one cell embryos (32, Table 2). Although hormones generally work at low concentrations, the growth of the genotypes from the transgenic lines obtained with our different tiGH-containing chimeric constructs will have to be compared. Since we do not know the optimum constitutive GH levels required for better growth increase in tilapia, results will have to be obtained empirically comparing different constructs and transgenic lines in heterozygous and homozygous animals, characterizing the tissues and levels of ectopic tiGH expression. Characterization of the "albino" Transgenic Tilapia Line With the CMV>tiGH>CAT>SV40 construct (CMV-tiGH in Table 2), a transgenic animal containing 1 copy of the transgene per cell was selected to establish a transgenic line (F0-3 in reference 37 and later designated as "albino") (32, 33). The transgene was stably transmitted to F1, F2 and F3 generations in a Mendelian fashion (Table 3). Ectopic, low level expression of tiGH was detected in gonad and muscle cells of F1 transgenic tilapia by in situ hybridization and immunohystochemical analysis of tissue sections (33 and data not shown). Nine months old transgenic F1 progeny were 82 % larger than the non-transgenic ones at p = 0.001 (Table 4). Under different culture conditions, 4 and 7 months old F2 transgenic fish were 37 % and 55 % larger than non-transgenic siblings at p = 0.01 and p = 0.009, respectively. Transgenic F2 and non-transgenic siblings were compared with respect to feeding motivation (FM), dominance status (DS), 50 % of sea water adaptation (SWA) and digestibility coefficient (Guillen, et al. manuscript in preparation). Transgenic tilapia showed a higher FM (p = 0.03) and DS (p = 0.0004) than non- transgenic siblings. Transgenic F2 tilapia also showed a better adaptability to sea water, suggesting that GH could be involved in osmotic regulation in this specie. No abnormalities have been recorded in this transgenic line. These results showed that low level ectopic expression of tiGH resulted in a growth acceleration in this transgenic tilapia line.
--------------------------------------------------------------
Table 3. Germ-line transmission of CMV-tiGH transgene in
the transgenic "albino" tilapia line.
--------------------------------------------------------------
Generation Number of animals Inheritance
(transgenic / non-transgenic)
--------------------------------------------------------------
P1 1 (1 / 0) -----
F1 (P1xWt) 38 (18 / 38) 47
F2 (F1xF1) 10 (8 / 10) 80
F3 (F2^+/-xF2^+/-) 10 (7 / 10) 70
--------------------------------------------------------------
Table 4. Characterization of F1 progeny derived from
transgenic "albino" tilapia.
--------------------------------------------------------------
Transgenic^a Non-transgenic^a
N 18 20
--------------------------------------------------------------
% inheritance 47 --
Mean weight+/-SE(g) 399.1+/-94.1 219.5+/-49.1
Weight range of
the males (g) 219.2-710.0^b, 95.9-472.1^d
Larger than the
largest control 44 % (8/18 --
Smaller than the
smallest control 0 --
Mean growth increase^e
(delta average weight
/delta ) 50.4+/-4.2 28.5+/-2.5
% difference 81.8^f --
^a Values for 9 months old tilapia in June 1994.
^b 75 % of transgenic males were larger than the largest
control.
^c N = 12, ^dN = 6.
^e Mean+/-SD.
^f Transgenic progeny were larger than non-transgenic progeny
at p = 0.001.
--------------------------------------------------------------
Ecological and Regulatory Issues associated with Transgenic Fish and Shellfish Consideration of the Safety/Risks Relation of Working with Transgenic Fish and Shellfish Although transgenic tilapia have been manipulated to accelerate growth, many characteristics of the parental organism should be maintained. We have not seen any abnormalities in transgenic tilapia, including analysis of homozygous animals. Food consumption, ecological impact and other aspects of these transgenic tilapia strains will have to be assayed under experimental conditions before introducing this technology into the national aquaculture programs. The current knowledge does not allow us to predict the full range or magnitude of phenotypic changes in particular lines of transgenic fish. In the case of the transfer of GHs, impact pathways on the ecosystem could include the following (39): The large size of individuals bearing introduced GH genes might favor them in agonistic interactions over food resources. Because GHs directly and indirectly affect behavior, transfer of such genes could impact the successful execution of life history-related events such as migration, territoriality and mating. Altered resources or substrate uses could result if, through accelerated growth, predatory transgenic individuals exhibit a larger mouth gap and make use of new prey items. Therefore, there is a need for carefully evaluating the safety/risks relation in the research and introduction of transgenic fish and shellfish into national aquaculture programs. A recent experiment comparing the DS of transgenic F2 and wild type (those directly obtained from natural ponds and not grown under laboratory conditions) tilapia suggested that transgenic populations accidentally escaping to natural ecosystems would have less chance to survive (Guillen, et al. manuscript in preparation). Wild type tilapia showed a higher DS than both transgenic and non-transgenic siblings (p = 1.13 E-12, ANOVA Single Factor Test). These preliminary observations addressed some of the concerns discussed (39) for the introduction of transgenic fish into the national aquaculture programs. Performance Standards for Safely Conducting Research with Genetically Modified Fish and Shellfish To conduct studies outside the laboratory, we held an ad hoc committee meeting devoted to the analysis of the conditions for releasing tilapia with accelerated growth in Cuba. This committee concluded that, under the conditions found in Cuba, little or no effect on the natural population will occur as a result of the accidental escape of transgenic fish, mainly because these natural populations do not exist and most of the fish species found now in the country have been introduced. Nevertheless, the committee recommended to follow the final draft of the "Performance Standards for Safely Conducting Research with Genetically Modified Fish and Shellfish" (documents No. 95-01 and 95-02, reference 40) prepared and released on April 15, 1995 by The U.S. Department of Agriculture, the Agricultural Biotechnology Research Advisory Committee, and the Working Group on Aquatic Biotechnology and Environmental Safety. These standards, as they state in the overview, are accompanied by flowcharts that guide the assessment pathway in a way that allows to consider and implement the necessary measures to safely conduct the experiments with transgenic fish to accumulate the data needed to fully characterize these new fish strains. Our experiments have been planned and conducted following these recommendations (appendix 1). The Importance of the Public Opinion
The public must be informed about the possibilities and limitations of transgenic technology. In Cuba, where the population is well educated, many people are aware of what genetic engineering and gene transfer mean. In the last 15 years, Cuba has experimented a rapid and successful development in modern biotechnology (41, 42). This effort together with widespread information on biotechnology and related technologies (43) have helped the public opinion to become favorable to these technologies and confide in their safety and results. Public confidence in the results of performance tests and ecological risk analyses, in addition to the results themselves, will prove critical in determining whether transgenic fish and shellfish enter into the national aquaculture programs. The scientific community has a great responsibility in the conduction and evaluation of experiments with transgenic organisms and in the education of the people to increase public confidence and to avoid extreme attitudes against the development and use of transgenic animals.
This paper was presented and discussed at the Expert Committee (Drs. Julio Baisre, Teresa Sanchez, Jose Vazquez, Zenaida Arboleya, Domingo Fonticiella, Jose Toledo, Emma Fajer, Adela Prieto, Jose de la Fuente, Oscar Hernandez, Rebeca Martinez, Isabel Guillen, Mario P. Estrada, Alina Rodriguez and Ricardo Lleonart) Meeting "Evaluation and risk analysis of the introduction of transgenic tilapia in Cuban aquaculture" organized by Centro de Ingenieria Genetica y Biotecnologia (CIGB), Centro de Investigaciones Pesqueras and the Ministry of Fisheries and held in the CIGB, Camaguey, Cuba in May 30 - June 2, 1995. We are indebted to Ivonne Blanco, Ileana Gonzalez and Yurima Hidalgo for assistance during the meeting and in the preparation of the manuscript. We are grateful to the staff of the Mammalian Cell Genetics Division of the CIGB for their support and fruitful discussions. This work was partially supported by the International Centre for Genetic Engineering and Biotechnology Collaborative Research Programme (project CRP/ CUB93-05).
References 1 International Marine Biotechnology Conference. Stouffer Harborplace Hotel. Baltimore. Maryland USA, October 13-16. 1991. 2 International Marine Biotechnology Conference. Tromso, Norway. 1994. 3 Gallardo N, Gonzalez R, Carrillo O, Wormhoudt A Van. Hormona de Crecimiento (GH) y Gastrina / Colecistoqui-nina (G/CCK) inmunoreactivas aisladas de Pectem maximus y utilizadas como factores de crecimiento. Advances in Modern Biotechnology 1992(a);1:18.7. 4 Gallardo N, Gonzalez R, Carrillo O, Rodriguez J, Arnanz C, Gonzalez R. Deteccion de Insulina y Hormona de Crecimiento inmunorreactiva en hepatopancreas de langosta (Panulirus argus) mediante radioinmunoanalisis. Advances in Modern Biotechnology 1992(b);1:18.8. 5 Hosoi N, Sekine S, Itoh S. Process for producing fish growth hormone polypeptide. Patent Number EPO 0239 075. 1988. 6 Itoh S, Tabe T, Ueda T, Kawauchi H. Method for culturing fish and sellfish using growth hormones as a feed suplement or on a water insoluble carrier. Patent Number EPO 0213 357. 1987. 7 Rentier-Delrue F, Martial J, Renard J. E. coli- derived rtiGH and/or rcfGH as food aditive for fish in an efficient amount such as 1 g to 1 mg per g of fish food per day with promoting growth and/or osmoregulation properties. Patent Number EPO 0377 540. 1990(a). 8 Rentier-Delrue F, Martial J, Renard A. Tilapia (O. niloticus) GH and Prolactin genes. Patent Number EPO 0387- 457. 1990(b). 9 Sakata S, Kawauchi H, Ono M. GH-like proteins from pituitary gland of fishes belonging to the order pleuronectina, the family Paralichthydae. Patent Number EPO 0443-790. 1991.
10 Smal J, Renard A. sGH for seawater adaptation of farmed salmonids. Patent Number EPO 0438-929. 1991. 11 Cabrera E, Pimentel R, Abad Z, Pina JC, Hernandez O, Lleonart R, de la Fuente J. Transient transformation of shrimp (Penaeus shmitti) embryos by DNA microinjection. Theriogenology 1995;43:180.
12 Hernandez O, Theron MC, Puissant C, Bearzotti M, Atlal J, Lebail PY, Cabrera E, Lleonart R, de la Fuente J, Houdebine LM. Effect of intervening sequences in the transient expression of tilapia growth hormone in mammalian and fish cells. Biotecnologia Aplicada / 1993(a);10:158-161.
13 Hernandez O, Attal J, Theron MC, Puissant C, Houdebine LM. Efficiency of introns from various origins in fish cells. Molecular Marine Biology and Biotechnology 1993(b);2(3):181- 188.
14 Lleonart R, Martinez R, Garcia del Barco D, Hernandez O, Castro FO, de la Fuente J. Reporter genes for in vivo transient gene expression studies in tilapia (Oreochromis aureus) and common carp (Cyprinus carpio) one celled embryos. Theriogenology 1994;41:240. 15 Garcia del Barco D, Martinez R, Hernandez O, Lleonart R, de la Fuente J. Differences in the transient expression directed by heterologous promoter and enhancer sequences in fish cells and embryos. Journal of Marine Biotechnology 1994;1:203- 205. 16 Sanchez T, Ponce de Leon R, Aguilar M, Vazquez J, McAndrew B. Response to selection and heritability for weight in Oreochromis aureus Steindachner after five generations of selection. Fifth International Symposium on Genetics in Aquaculture, Dalhousie University, Halifax, Nova Scotia, Canada, p. 126 (Abstract). 1994. 17 De la Fuente J, Hernandez O, Guillen I, Castro FO, Aguilar A, Herrera L, Liver CU, Perez A. Transgenesis en peces y su aplicacion en Biotecnologia. Biotecnologia Aplicada 1991; 8:123-139. 18 Godman RM, Hauptli H, Crossway A, Knauf VC. Gene transfer in crop improvement. Science 1987;236:48-54. 19 Pursel VG, Pinkert CA, Miller KF, Boit DJ, Campbell RG, Palmiter RD, Brinster RL, Hammer RE. Genetic engineering of livestock. Science 1989; 244:1281-1288. 20 Powers DA. Fish as model systems. Science 1989;246:352- 358.
21 Hew CL, Gong Z. Transgenic fish. A new technology for fish biology and aquaculture. Biology International 1992; 24:2- 10.
22 Maclean N, Rahman A. Transgenic Fish. In: Animals with novel genes. (N. Maclean.ed). Cambridge University Press, Australia. 1994;63-105. 23 Zhu Z, Li G, He L, Chen S. Novel gene transfer into the fertilized eggs of gold fish (Carassius auratus L.1758). Angew Ichthyol 1985;1:31-34. 24 Zhu Z, Xu K, Li G, Xie Y, He L. Biological effects of human growth hormone gene microinjected into the fertilized eggs of loach Misgurnus anguillicaudatus (Cantor). Kexue Tongbao 1986; 31:988-990. 25 Zhu Z. Generation of fast growing transgenic fish: Methods and Mechanisms. In: Transgenic Fish. Hew CL and Fletcher (eds.) World Scientific Publishing Co., Singapore 1991;92- 119. 26 Zhang P, Hayat M, Joyce C, Gonzalez-Villasenor LI, Lin CM, Dunham RA, Chen TT, Powers DA. Gene transfer expression and inheritance of PRSV-rainbow trout-GH cDNA in the common carp, Cyprinus carpio (L). Molecular Reproduction and Development 1990; 25:3-13. 27 Chen TT, Lin CM, Dunham RA, Powers DA. Integration, expression and inheritance of foreign fish growth homone gene in transgenic fish. In: Transgenic Fish. C.L.Hew and Fletcher (eds.). World Scientific Publishing Co., Singapore 1992 pp: 164-175. 28 Du SJ, Gong Z, Fletcher GL, Sears MA, King MJ, Idler DR, Hew CL. Dramatic growth enhancement in transgenic Atlantic salmon: use of an all fish chimeric growth hormone gene construct. BioTechnology 1992;10:176-181. 29 Guise KS, Hackett PB, Faras AJ. Transfer of genes encoding neomycin resistance, chloramphenicol acetyl transferase, and growth hormone into goldfish and northern pike. In: Transgenic Fish. CL Hew and Fletcher (eds.). World Scientific Publishing Co., Singapore 1992 pp: 142-164. 30 Lu JK, Chrisman CL, Chen TT. Integration and germline transmission of human growth hormone gene in medaka (Oryzias latipes). In Second Marine Biotechnology Conference, Baltimore MD 1991;77 (abstract). 31 Devlin RH, Yesaki TY, Biagi CA, Donaldson EM. Extraordinary salmon growth Nature 1994;371:209-210. 32 De la Fuente J, Martinez R, Estrada MP, Hernandez O, Cabrera E, Garcia del Barco D, Lleonart R, Pimentel R, Morales R, Herrera F, Morales A, Guillen I, Pina JC. Towards growth manipulation in tilapia (Oreochromis sp.): generation of transgenic tilapia with chimeric constructs containing the tilapia growth hormone cDNA. Journal of Marine Biotechnology 1995;3:216-219. 33 Martinez R, Estrada MP, Berlanga J, Guillen I, Hernandez O, Cabrera E, Pimentel R, Morales R, Herrera F, Morales A, Pina JC, Abad Z, Sanchez V, Melamed P, Lleonart R, de la Fuente J. Growth enhancement in transgenic tilapia by ectopic expression of tilapia growth hormone. Molecular Marine Bioloy and Biotechnology 1996;5(1):62-70. 34 Brem G, Brening B, Horstgen-Schwark G, Winnacker EL. Gene Transfer in Tilapia (Oreochromis niloticus). Aquaculture 1988;68:209-219. 35 Rahman MA, Maclean N. Production of transgenic tilapia (Oreochromis niloticus) by one cell stage microinjection. Aquaculture 1992;105:209-219. 36 Phillips CP, Cohler CC, Muhlach W. Procedural protocol, survival to hatching and plasmid DNA fate after microinjection into tilapia zygotes. J World Aqua Soc 1992;23(2):98-113. 37 Martinez R, Garcia del Barco D, Hernandez O, Lleonart R, Herrera F, Cabrera E, de la Fuente J. Generation of transgenic tilapia with a quimeric gene that directs the synthesis of mRNA in CHO cells. Miami Short Reports (Ed. By Whelan WJ, Ahmad F, Bialy H, Black S, Lou King M, Rabin MB, Solomonson LP and Vasil LIK) 1992;2:68. 38 Lleonart R, Garcia del Barco D, Hernandez O, de la Fuente J. Microinjection of DNA into tilapia (Oreochromis aureus) and common carp (Cyprinus carpio) one cell embryos: a novel system for in vivo transient gene expression studies. Advances in Modern Biotechnology 1992;1:18.1. 39 Hallerman EM, Kapuscinski AR. Ecological and regulatory uncertainties associated with transgenic fish. In: Transgenic Fish. Hew CL and Fletcher (eds.). World Scientific Publishing Co., Singapore. 1992 pp: 209-228. 40 Department of Agriculture, the Agricultural Biotechnology Research Advisory Committee, and the Working Group on Aquatic Biotechnology and Environmental Safety. Performance Standars for Safety Conducting Research with Genetically Modified Fish and Shellfish. Article 21,753 of the Minnesota Agricultural Experiment Station Scientific Journal Series. 1995. 41 Robinson C. Biotechnology in Cuba: tackling Third-World problems with front line-tehcnology. TIBTECH 1993;11:80-84.
42 Bialy H. Cuba s Biotechnology and The United States Embargo. BioTechnology 1995;13:288.
43 Hernandez Serrano L. Obtencion de animales transgenicos en Cuba. Juventud Rebelde, 23 de abril 1989. Appendix Worksheet Accompanying Performance Standards for Safely Conducting Research with Genetically Modified Finfish and Shellfish Introduction The Performance Standards for Safely Conducting Research with Genetically Modified Finfish and Shellfish are voluntary guidelines intended to aid researchers and institutions in assessing the genetic and ecological effects of research activities involving genetically modified fish, crustaceans, and molluscs, and in determining appropriate procedures and safeguards so that the research can be conducted without causing adverse impacts on the environment. The Flowcharts of the Performance Standards guide researchers in identifying, assessing and managing specific risks. This Worksheet accompanies the Flowcharts. Once completed by the researcher, the Worksheet will document both the decision path taken through the flowcharts of the Performance Standards, and any risk management measures. It is designed to assist researchers and reviewers in evaluating the project. Until the Performance Standards are incorporated into a computerized expert system with the capability of producing a hard-copy trace of the decision path, this worksheet should be used. Principal researcher: Jose de la Fuente
Proposed Project Field Testing of Transgenic Tilapia Expressing the Tilapia Growth Hormone cDNA in Cuba. Please mark your response to a question by checking "Yes," "No," "Don't know," "EXIT," or by indicating your routing to a subsequent flowchart. The marking of more than one blank may be appropriate in particular situations. Attach written explanatory material as directed below.
Flowchart Documentation
Please list the number of all flowcharts that you used:
---------------------------------------------------------------------
--x-- Consult Appendix B.
----- EXIT the standards. Attach your rationale. ----- No. Continue at flowchart II.B.
--x-- IV.A. in Ecosystem effects assessment.
----- VI.B. Risk management-insufficient information. ----- EXIT the standards. Attach your rationale. ----- EXIT to Aquatic Nuisance Species Protocol (Appendix A)
----- II.C. Assess impact of additional modifications. ----- EXIT the standards. Attach your rationale. ----- No. Continue at flowchart II.C.
----- EXIT the standard. Attach your rationale. ----- EXIT Aquatic Nuisance Species Protocol (see Appendix A).
----- II.C.1. Assess potential impact of interspecific hybridization. ----- EXIT the Standards. Attach your rationale. ----- No. EXIT the standards. Attach your rationale.
Attach a written description of any identified risks.
----- VI.A. Risk management-specific risks: Manage risks to protected population. ----- VI.A. Risk management-specific risks: Manage risks of losing population of pure species. ----- EXIT the standards. Attach your rationale. ----- EXIT to Aquatic Nuisance Species Protocol.
----- VI.A. Risk management-specific risks. Manage risks to protected populations. ----- EXIT the standards. Attach your rationale.
----- VI.B. Risk management-insufficient information. ----- EXIT the standards. Attach your rationale.
----- VI.A. Risk management-specific risks. Manage risk of decline in population abundance. --x-- VI.B. Risk management- insufficient information.
----- EXIT the standards. Attach your rationale.
----- VI.B. Risk management - insufficient information. ----- EXIT the standards. Attach your rationale.
----- VI.B. Risk management-insufficient information. ----- EXIT the standards. Attach your rationale.
----- VI.A. Risk management-specific risks. Manage risks of alteration of ecosystem processes. ----- VI.B. Risk management-insufficient information. ----- EXIT the standards. Attach your rationale.
VI.B. If you were directed to use the flowchart regarding risk management when there is insufficient information to assess risks, what measures do you plan to adopt to effectively confine the proposed experiment? Attach a written description of the risk management measures you plan to implement.
Be certain to address the topics listed in the Risk Management Documentation section below.
Additional Questions 1. Are you working with a non-indigenous species?
--x-- No.
See the list of experts in acknowledgements.
June 2, 1995
----------------------- -------------
Signature of researcher Date
Address and Phone No. Division of Mammalian Cell Genetics. Centro de
Ingenieria Genetica y Biotecnologia. P.O. Box 6162. Havana 6, CUBAPhone No.: 21 6221 Fax No.: 218070 E-mail: invest@serverdos.cigb.edu.cu
Risk Management Documentation As part of the compliance with the voluntary Performance Standards, the researcher must describe and provide the rationale for the risk management measures. Major points explained in the text on Risk Management Recommendations are listed below. Researchers and reviewers should read the text on Risk Management Recommendations before using this portion of the Worksheet. The risk management documentation must fully respond to these major points. For items which request a narrative response, attach your written responses and identify the numbered item being addressed.
Project Setting 1. Explain how the setting and structures of the project prevent accidental releases during flooding or other natural disasters. A. If project involves placement of GMOs in uncovered outside tanks or ponds, is there the potential for sudden high winds to wash organisms into a natural water body (accessible ecosystem) via water spray or waves?
--x-- No. Proceed to item 2.
Design of Barriers The Standards identify four types of barriers: (1) physical or chemical; (2) mechanical; (3) biological and (4) scale of experiment as a barrier. 2. Was the project site chosen because the surrounding accessible ecosystems are lethal to all life stages of the GMO?
--x-- No. Proceed to item 3.
(b) Explain how the setting reduces the need for barriers on- site.
Yes b. Effluent and drawdown water? (Note: if discharge to sanitary sewer is used as one barrier against accidental escape of GMOs in effluent, at least one additional barrier is necessary) No c. Waste slurries? No d. Disposal of experimental animals? No e. Aerosols (applies only to shellfish with small larvae)? No f. Equipment cleaning and storage?
5. For each escape path identified in items 3 and 4 above, describe the arrangement and types of barriers to escape; a diagram of layout of barriers at the site or facility may be useful. Describe: treatment and disposal of waste slurries; disposal of experimental animals; and cleaning and storage of equipment. 6. Describe how the types and numbers of barriers in series are sufficient to achieve the "acceptable number of accidental escapees" specified in Flowcharts VI.A. or VI.B.
Special Concerns 7. Yes If biological barriers are used for a given escape path, does the path have at least one other type of barrier? (Because of their variable efficacy, biological barriers cannot comprise the entire set of barriers). 8. n.a. If scale is used as a barrier, are you certain the GMO is not a self-fertilizing hemaphrodite or true parthenogen? Attach supporting evidence.
Security 9. Describe the security measure implemented to:
b. Prevent unauthorized access to the site, and c. Eliminate access for predators who could potentially carry animals off-site (applies only to outdoor projects).
10. Describe and justify the adequacy of the entire set of installed alarms. Be sure to address the following:
b. Do all installed alarms have backup power? c. Describe the plan for notifying designated personnel.
Operational Plan 11. Attach the written operational plan. Required components are:
b. Traffic Control. c. Record Keeping. d. Emergency Response Plan.
Review and Inspection 12. Has your institutional biosafety committee, biosafety officer, or other appropriate expert reviewed and approved the proposed project and its risk management measures? If not, explain the review status of your project.
----- No
----- No
----- pending ----- not yet submitted
Worksheet Accompanying Performance Standards for Safely Conducting Research with Genetically Modified Finfish and Shellfish.
Proposed Project Field testing of transgenic tilapia expressing the tilapia growth hormone cDNA in Cuba. Summary of the Risks Identified and Proposed Measures
Risks identified: The accessible ecosystems contain co-specific species with which transgenic tilapia could breed. Since transgenic aninals are fertile, there is a risk for a potential introgression of the chimeric tilapia growth hormone gene into natural populations. Nevertheless, in the case of Cuba, tilapia were introduced in the sixties and there is no risk of affecting endogenous species. We are dealing with ecosystems deliberately modified. However, because we do not have all the information, we decided to evaluate the reproductive potential, genetic flow and behavior of transgenic tilapia while preventing the accidental escape of these genetically modified organisms. Documentation for risk management: The proyect will be conducted in artificial ponds in G and M in the province La Habana. Both sites guarantee the safety required for the experiment (eg. location above the 100 year flood level). G: This site possesses artificial ponds of 6 and 140 m^2. Effluents and drawdown water discharge directly to the ocean through a 4 Km canal. No other surface water bodies are located in the area. Mechanical, chemical and biological barriers were placed at the initial and final points of the water system of the project. This site will be used for the conformation of the reproduction stock and for the evaluation of the industrial process T^+/+ x wt. M: This site possesses artificial ponds of 0.4, 0.8 and 1 ha. The water comes through gravity from a large artificial water body and the effluents and drawdown water discharge directly to the ocean through a 5 Km canal. Mechanical barriers were placed at the initial and final points of the water system of the project. The 0.4 ha pond will be used in a second part of the project for the evaluation of transgenic heterozygous tilapia (T^+/+ x wt) under extensive culture conditions. Intensive culture conditions will be evaluated under controlled laboratory conditions. In both sites the authorized personnel involved in the project will be controlled to prevent unauthorized access to the site. Security guards and other security measures will be also implemented. Copyright 1996 Elfos Scientiae The following images related to this document are available:Line drawing images[ba96098a.gif] |
| |||||||||
