Progress in the evaluation of transgenic fish for possible ecological risk and its containment strategies

Genetically improved transgenic fish possess many beneficial economic traits; however, the commercial aquaculture of transgenic fish has not been performed till date. One of the major reasons for this is the possible ecological risk associated with the escape or release of the transgenic fish. Using a growth hormone transgenic fish with rapid growth characteristics as a subject, this paper analyzes the following: the essence of the potential ecological risks posed by transgenic fish; ecological risk in the current situation due to transgenic fish via one-factor phenotypic and fitness analysis, and mathematical model deduction. Then, it expounds new ideas and the latest findings using an artificially simulated ecosystem for the evaluation of the ecological risks posed by transgenic fish. Further, the study comments on the strategies and principles of controlling these ecological risks by using a triplold approach. Based on these results, we propose that ecological risk evaluation and prevention strategies are indispensable important components and should be accompanied with breeding research in order to provide enlightments for transgenic fish breeding, evaluation of the ecological risks posed by transgenic fish, and development of containment strategies against the risks.

Antisense for gonadotropin-releasing hormone reduces gonadotropin synthesis and gonadal development in transgenic common carp (Cyprinus carpio)

Generating transgenic fish with desirable traits (e.g., rapid growth, larger size, etc.) for commercial use has been hampered by concerns for biosafety and competition if these fish are released into the environment. These obstacles may be overcome by producing transgenic fish that are sterile, possibly by inhibiting hormones related to reproduction. In vertebrates, synthesis and release of gonadotropin (GtH) and other reproductive hormones is mediated by gonadotropin-releasing hormone (GnRH). Recently two cDNA sequences encoding salmon-type GnRH (sGnRH) decapeptides were cloned from common carp (Cyprinus carpio). This study analyzed the expression of these two genes using real-time polymerase chain reaction (RT-PCR) in different tissues carp at varying developmental stages. Transcripts of both genes were detected in ovary and testis in mature and regressed, but not in juvenile carp. To evaluate the effects of sGnRH inhibition, the recombinant gene CAsGnRHpc-antisense, expressing antisense sGnRH RNA driven by a carp beta-actin promoter, was constructed. Blocking sGnRH expression using antisense sGnRH significantly decreased GtH in the blood of male transgenic carp. Furthermore, some antisense transgenic fish had no gonadal development and were completely sterile. These data demonstrate that sGnRH is important for GtH synthesis and development of reproductive organs in carp. Also, the antisense sGnRH strategy may prove effective in generating sterile transgenic fish, eliminating environmental concerns these fish may raise. (c) 2007 Published by Elsevier B.V.

Site-directed gene integration in transgenic zebrafish mediated by cre recombinase using a combination of mutant Lox sites

With current gene-transfer techniques in fish, insertion of DNA into the genome occurs randomly and in many instances at multiple sites. Associated position effects, copy number differences, and multiple gene interactions make gene expression experiments difficult to interpret and fish phenotype less predictable. To meet different fish engineering needs, we describe here a gene targeting model in zebrafish. At first, four target zebrafish lines, each harboring a single genomic lox71 target site, were generated by zebrafish transgenesis. The zygotes of transgenic zebrafish lines were coinjected with capped Cre mRNA and a knockin vector pZklox66RFP. Site-specific integration event happened from one target zebrafish line. In this line two integrant zebrafish were obtained from more than 80,000 targeted embryos (integrating efficiency about 10(-4) to 10(-5)) and confirmed to have a sole copy of the integrating DNA at the target genome site. Genomic polymerase chain reaction analysis and DNA sequencing verified the correct gene target events where lox71 and lox66 have accurately recombined into double mutant lox72 and wild-type loxP. Each integrant zebrafish chosen for analysis harbored the transgene rfp at the designated egfp concatenates. Although the Cre-mediated recombination is site specific, it is dependent on a randomly placed target site. That is, a genomic target cannot be preselected for integration based solely on its sequence. Conclusively, an rfp reporter gene was successfully inserted into the egfp target locus of zebrafish genome by Cre-lox-mediated recombination. This site-directed knockin system using the lox71/lox66 combination should be a promising gene-targeting platform serving various purposes in fish genetic engineering.

Rapid growth cost in “all-fish” growth hormone gene transgenic carp: Reduced critical swimming speed

Evidence has accumulated that there is a trade-off between benefits and costs associated with rapid growth. A trade-off between growth rates and critical. swimming speed (U-crit) had been also reported to be common in teleost fish. We hypothesize that growth acceleration in the F-3 generation of "all-fish" growth hormone gene (GH) transgenic common carp (Cyprinus carpio L.) would reduce the swimming abilities. Growth and swimming performance between transgenic fish and non-transgenic controls were) compared. The results showed that transgenic fish had a mean body weight 1.4-1.9-fold heavier, and a mean specific growth rate (SGR) value 6%-10% higher than the controls. Transgenic fish, however, had a mean absolute U-crit (cm/s) value 22% or mean relative Ucrit (BL/s) value 24% lower than the controls. It suggested that fast-growing "all-fish" GH-transgenic carp were inferior swimmers. It is also supported that there was a trade-off between growth rates and swimming performance, i.e. faster-growing individuals had lower critical swimming speed.

Fish ES cells and applications to biotechnology

ES cells provide a promising tool for the generation of transgenic animals with site-directed mutations. When ES cells colonize germ cells in chimeras, transgenic animals with modified phenotypes are generated and used either for functional genomics studies or for improving productivity in commercial settings. Althought the ES cell approach has been limited to, mice, there is strong interest for developing the technology in fish.. We describe the step-by-step procedure for developing ES cells in fish. Key aspects include avoiding cell differentiation, specific in vitro traits of pluripotency, and, most importantly, testing for production of chimeric animals as the main evidence of pluripotency. The entire process focuses on two model species, zebrafish and medaka, in which most work has been done. The achievements attained in these species, as well as their applicability to other commercial fish, are discussed. Because of the difficulties relating to germ line competence, mostly of long-term fish ES cells, alternative cell-based approaches such as primordial germ cells and nuclear transfer need to be considered. Although progress to date has been slow, there are promising achievements in homologous recombination and alternative avenues yet to be explored that can bring ES technology in fish to fruition.

Growth and energy budget of F-2 'all-fish' growth hormone gene transgenic common carp

The growth and energy budget for F-2 'all-fish' growth hormone gene transgenic common carp Cyprinus carpio of two body sizes were investigated at 29.2 degrees C for 21 days. Specific growth rate, feed intake, feed efficiency, digestibility coefficients of dry matter and protein, gross energy intake (I-E), and the proportion of I-E utilized for heat production (H-E) were significantly higher in the transgenics than in the controls. The proportion of I-E directed to waste products [faecal energy (F-E) and excretory energy loss (Z(E) + U-E) where Z(E) is through the gills and U-E through the kidney], and the proportion of metabolizable energy (M-E) for recovered energy (R-E) were significantly lower in the transgenics than in the controls. The average energy budget equation of transgenic fish was as follows: 100 I-E = 19.3 F-E + 6.0 (Z(E) + U-E) + 45.2 H-E + 29.5 R-E or 100 M-E = 60.5 H-E + 39.5 R-E. The average energy budget equation of the controls was: 100 I-E = 25.2 F-E + 7.4 (Z(E) + U-E) + 35.5 H-E + 31.9 R-E or 100 M-E = 52.7 H-E + 47.3 R-E. These findings indicate that the high growth rate of 'all-fish' transgenic common carp relative to their non-transgenic counterparts was due to their increased feed intake, reduced lose of waste productions and improved feed efficiency. The benefit of the increased energy intake by transgenic fish, however, was diminished by their increased metabolism.

Developments in transgenic fish in the people's Republic of China

In the People's Republic of China, genetically modified (GM) fish are being developed primarily to produce desirable alterations to growth rates or feed-conversion efficiency. Up to the present, no transgenic fish have been commercially approved for human consumption. This review introduces advances in the People's Republic of China in transgenic fish studies, biosafety studies of fast-growth GM fish, and the regulation of GM fish.

Characterization of transgene integration pattern in F4 hGH-transgenic common carp (Cyprinus carpio L.)

The integration pattern and adjacent host sequences of the inserted pMThGH-transgene in the F4 hGH-transgenic common carp were extensively studied. Here we show that each F4 transgenic fish contained about 200 copies of the pMThGH-transgene and the transgenes were integrated into the host genome generally with concatemers in a head-to-tail arrangement at 4-5 insertion sites. By using a method of plasmid rescue, four hundred copies of transgenes from two individuals of F4 transgenic fish, A and B, were recovered and clarified into 6 classes. All classes of recovered transgenes contained either complete or partial pMThGH sequences. The class I, which comprised 83% and 84.5% respectively of the recovered transgene copies from fish A and B, had maintained the original configuration, indicating that most transgenes were faithfully inherited during the four generations of reproduction. The other five classes were different from the original configuration in both molecular weight and restriction map, indicating that a few transgenes had undergone mutation, rearrangement or deletion during integration and germline transmission. In the five types of aberrant transgenes, three flanking sequences of the host genome were analyzed. These sequences were common carp beta-actin gene, common carp DNA sequences homologous to mouse phosphoglycerate kinase-1 and human epidermal keratin 14, respectively.

Cytoplasmic impact on cross-genus cloned fish derived from transgenic common carp (Cyprinus carpio) nuclei and goldfish (Carassius auratus) enucleated eggs

In previous studies of nuclear transplantation, most cloned animals were obtained by intraspecies nuclear transfer and are phenotypically identical to their nuclear donors; furthermore, there was no further report on successful fish cloning since the report of cloned zebrafish. Here we report the production of seven cross-genus cloned fish by transferring nuclei from transgenic common carp into enucleated eggs of goldfish. Nuclear genomes of the cloned fish were exclusively derived from the nuclear donor species, common carp, whereas the mitochondrial DNA from the donor carp gradually disappeared during the development of nuclear transfer (NT) embryos. The somite development process and somite number of nuclear transplants were consistent with the recipient species, goldfish, rather than the nuclear donor species, common carp. This resulted in a long-lasting effect on the vertebral numbers of the cloned fish, which belonged to the range of goldfish. These demonstrate that fish egg cytoplasm not only can support the development driven by transplanted nuclei from a distantly related species at the genus scale but also can modulate development of the nuclear transplants.

Enhanced resistance to Aeromonas hydrophila infection and enhanced phagocytic activities in human lactoferrin-transgenic grass carp (Ctenopharyngodon idellus)

Human lactoferrin (hLF) is an iron-binding protein with antimicrobial and immunomodulatory activities. hLF cDNA was transferred into grass carp via electroporated sperm. The production of transgenic fish was as high as 55% tinder the best parameters. 2(11) pulses and 20-min incubation. The expression of the transgene was demonstrated by the detection of hLF mRNA by RT-PCR. We also investigated the response of G(0) transgenic grass carp to Aeromonas hydrophila infection. Serum lysozyme activities (P>0.05) and phagocytic activities of kidney cells (P<0.05) were measured in transgenic individuals. The transgenic fish not only cleared A. hydrophila significantly faster than the control carp (P<0.05), but also showed enhanced phagocytic activities. The result shows that hLF has immunomodulatory activities in hLF-transgenic grass carp. The transgenic grass carp exhibited enhanced immunity to A. hydrophila infection. These results reveal that the mechanisms of disease resistance are different between hLF-transgenic plants and hLF-transgenic grass carp. (C) 2004 Elsevier B.V. All rights reserved.

Transgenes in F-4 pMThGH-transgenic common carp (Cyprinus carpio L.) are highly polymorphic

To gain information on the integration pattern of pMThGH-transgene, 50 transgenes were recovered from F-4 generation of pMThGH transgenic common carp (Cyprinus carpio L,) and 33 recovered genes were analyzed. The restriction maps of these recovered genes were constructed by digestion with five kinds of enzymes. These transgenes can be classified into 4 types according to their restriction maps. Only one type of transgenes maintains its original molecular form, whereas the other three types are very different from the original one and vary each other on both molecular weight and restriction maps. This implies that the sequences of most transgenes have been deleted and/or rearranged during integration and inheritance. The results of PCR amplification and Southern blot hybridization indicate that MThGH in Type I transgene keeps intact but most of its sequence has been deleted in other three types. All these results suggest that transgenes in F-4 generation of transgenic carp are highly polymorphic. Two DNA fragments concerning integration site of transgenes were cloned from recovered transgenes, and found to be homologous to the 5'UTR of beta -actin gene of common carp and mouse mRNA for receptor tyrosine kinase (RTK), respectively.

Nuclear transplantation with early-embryonic cells of transgenic fish

Like other transgenic animals, transgenic fishes produced by microinjection are transgenic mosaics. In order to produce homogenous transgenic fish, the transgenic blastula or gastrula cells were dissociated from Carassius auratus, Pengze var, and Cyprinus carpio, Huanghe var., and the nuclei were transferred into the mature eggs of the same species via microinjection or electro-fusion. Five nuclear-transferred Carassius auratus, Pengze var. and one Cyprinus carpio, Huanghe var. were obtained and the existence of the transgene was detected. The possibility of generating homogenous strain of transgenic fish by nuclear transplantation with transgenic early-embryonic cells is discussed.

Whole-body amino acid pattern of F-4 human growth hormone gene-transgenic red common carp (Cyprinus carpio) fed diets with different protein levels

F-4 generation of human growth hormone (hGH) gene-transgenic red common carp, and the non-transgenic controls were fed for 8 weeks on purified diets with 20%, 30% or 40% protein. Analysis of whole-body amino acids showed that the proportions of lysine, leucine, phenylalanine, valine and alanine, as percentages of body protein, increased significantly, while those of arginine, glutamic acid and tyrosine decreased, with increases in dietary protein level in at least one strain of fish. Proportions of the other amino acids were unaffected by the diets. The proportions of lysine and arginine were significantly higher, while those of leucine and alanine were lower in the transgenics than in the controls in at least one diet group. Proportions of the other amino acids were unaffected by strain. The results suggest that the whole-body amino acid profile of transgenic carp, when expressed as proportions of body protein, was in general, similar to that of the non-transgenic controls. (C) 2000 Elsevier Science B.V. All rights reserved.

Time course of foreign gene integration and expression in transgenic fish embryos

Using a nuclear transplantation approach, the integration and expression of the green fluorescent protein (GFP) gene in the embryogenesis of transgenic leach (Misgurnus anguillicaudatus Cantor) have been studied. The GFP gene expression is first observed at the gastrula stage, which is consistent with the initiation of cell differentiation of fish embryos. The time course of the foreign gene expression is correlated with the regulatory sequences. The expression efficiency also depends on the gene configuration: the expression of pre-integrating circular plasmid at early embryos is higher than that of the linear plasmid. The integration of the GFP gene is first detected at the blastula stage and lasts for quite a long period. When two types of different plasmids are co-injected into fertilized eggs, the behavior of their integration and expression is not identical.