The zebrafish as a model system in developmental, toxicological and transgenic research

The zebrafish has long been used as a model system in fisheries biology and toxicology. More recently, it has also become the focus of a major research effort into understanding the molecular and cellular events which dictate the development of vertebrate embryos. As well, the zebrafish has proven attractive in studies examining the factors which affect the creation of transgenic fish and the expression of transgenes. The advances which have been made in these areas have firmly established this small aquarium fish as a major model system in biological and biotechnological research.     

Transgene expression in zebrafish: A comparison of retroviral-vector and DNA-injection approaches.

To assess alternative methods for introducing expressing transgenes into the germ line of zebrafish, transgenic fish that express a nuclear-targeted, enhanced, green fluorescent protein (eGFP) gene were produced using both pseudotyped retroviral vector infection and DNA microinjection of embryos. Germ-line transgenic founders were identified and the embryonic progeny of these founders were evaluated for the extent and pattern of eGFP expression. To compare the two modes of transgenesis, both vectors used the Xenopus translational elongation factor 1-alpha enhancer/promoter regulatory cassette. Several transgenic founder fish which transferred eGFP expression to their progeny were identified. The gene expression patterns are described and compared for the two modes of gene transfer. Transient expression of eGFP was detected 1 day after introducing the transgenes via either DNA microinjection or retroviral vector infection. In both cases of gene transfer, transgenic females produced eGFP-positive progeny even before the zygotic genome was turned on. Therefore, GFP was being provided by the oocyte before fertilization. A transgenic female revealed eGFP expression in her ovarian follicles. The qualitative patterns of gene expression in the transgenic progeny embryos after zygotic induction of gene expression were similar and independent of the mode of transgenesis. The appearance of newly synthesized GFP is detectable within 5-7 h after fertilization. The variability of the extent of eGFP expression from transgenic founder to transgenic founder was wider for the DNA-injection transgenics than for the retroviral vector-produced transgenics. The ability to provide expressing germ-line transgenic progeny via retroviral vector infection provides both an alternative mode of transgenesis for zebrafish work and a possible means of easily assessing the insertional mutagenesis frequency of retroviral vector infection of zebrafish embryos. However, because of the transfer of GFP from oocyte to embryo, the stability of GFP may create problems of analysis in embryos which develop as quickly as those of zebrafish.     

Rearing in Seawater Mesocosms Improves the Spawning Performance of Growth Hormone Transgenic and Wild-Type Coho Salmon

Growth hormone (GH) transgenes can significantly accelerate growth rates in fish and cause associated alterations to their physiology and behaviour. Concern exists regarding potential environmental risks of GH transgenic fish, should they enter natural ecosystems. In particular, whether they can reproduce and generate viable offspring under natural conditions is poorly understood. In previous studies, GH transgenic salmon grown under contained culture conditions had lower spawning behaviour and reproductive success relative to wild-type fish reared in nature. However, wild-type salmon cultured in equal conditions also had limited reproductive success. As such, whether decreased reproductive success of GH transgenic salmon is due to the action of the transgene or to secondary effects of culture (or a combination) has not been fully ascertained. Hence, salmon were reared in large (350,000 L), semi-natural, seawater tanks (termed mesocosms) designed to minimize effects of standard laboratory culture conditions, and the reproductive success of wild-type and GH transgenic coho salmon from mesocosms were compared with that of wild-type fish from nature. Mesocosm rearing partially restored spawning behaviour and success of wild-type fish relative to culture rearing, but remained lower overall than those reared in nature. GH transgenic salmon reared in the mesocosm had similar spawning behaviour and success as wild-type fish reared in the mesocosm when in full competition and without competition, but had lower success in male-only competition experiments. There was evidence of genotype×environmental interactions on spawning success, so that spawning success of transgenic fish, should they escape to natural systems in early life, cannot be predicted with low uncertainty. Under the present conditions, we found no evidence to support enhanced mating capabilities of GH transgenic coho salmon compared to wild-type salmon. However, it is clear that GH transgenic salmon are capable of successful spawning, and can reproduce with wild-type fish from natural systems.     

In vivo activation of human immunodeficiency virus type 1 long terminal repeat by UV type A (UV-A) light plus psoralen and UV-B light in the skin of transgenic mice.

UV irradiation has been shown to activate the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in cell culture; however, only limited studies have been described in vivo. UV light has been categorized as UV-A (400 to 315 nm), -B (315 to 280 nm), or -C (less than 280 nm); the longer wavelengths are less harmful but more penetrative. Highly penetrative UV-A radiation constitutes the vast majority of UV sunlight reaching the earth's surface but is normally harmless. UV-B irradiation is more harmful but less prevalent than UV-A. In this report, the HIV-1 LTR-luciferase gene in the skin of transgenic mice was markedly activated when exposed to UV-B irradiation. The LTR in the skin of transgenic mice pretreated topically with a photosensitizing agent (psoralen) was also activated to similar levels when exposed to UV-A light. A 2-h exposure to sunlight activated the LTR in skin treated with psoralen, whereas the LTR in skin not treated with psoralen was activated after 7 h of sunlight exposure. The HIV-1 LTR-beta-galactosidase reporter gene was preferentially activated by UV-B irradiation in a small population of epidermal cells. The transgenic mouse models carrying HIV-1 LTR-luciferase and LTR-beta-galactosidase reporter genes have been used to demonstrate the in vivo UV-induced activation of the LTR and might be used to evaluate other environmental factors or pharmacologic substances that might potentially activate the HIV-1 LTR in vivo.     

New technology for an old favorite: lentiviral transgenesis and RNAi in rats

The ability to produce targeted deletions in the mouse genome via homologous recombination has been a hallmark of mouse genetics, and has lead to the production of thousands of gene knockouts. New technologies are making it possible to disrupt gene function in many other species. This article reviews some of these methods, highlighting the powerful combination of lentiviral vectors with RNA interference (RNAi), which allows one to produce transgenic animals expressing short hairpin RNA (shRNA) to “knock down” specific gene expression. Lentiviral transduction of embryos has been shown to be a highly efficient means of transgenesis, and is particularly promising for animals that are considered difficult to genetically modify by DNA pronuclear injection. This technique has been popular for introducing transgenes for shRNA expression into rodents and its utility for creating new genetic models has already been demonstrated. One of the purported advantages of in vivo RNAi is that shRNA expressing transgenes would be expected to act in a dominant nature, resulting in a phenotype in founder animals. However, one possible concern with lentiviral-mediated transgenesis is the potential for mosaicism in founders, and the data for this phenomenon and the potential causes and solutions are discussed. Emphasis is placed on the application of in vivo RNAi, and other reverse genetic methods, for creating new genetic models in the rat.     

Sub-chronic exposure to 1,1-dichloropropene induces frameshift mutations in lambda transgenic medaka

1,1-Dichloropropene (1,1-DCP) is a contaminant present in both ground and surface waters used as sources for drinking water. Structural similarity to several compounds with known mutagenicity and carcinogenicity, and recent demonstration of mutagenicity in vitro, suggest this compound may be similarly mutagenic in vivo. A transgenic fish model, the lamda transgenic medaka, was used to evaluate the potential mutagenicity of this contaminant in vivo following sub-chronic exposure for 6 weeks. Mutant frequencies of the cII target gene (MF) increased six-fold in the livers of fish exposed to the lowest 1,1-DCP exposure concentration (0.44 mg/L, MF = 18.4 x 10(-5), and increased with each treatment, culminating in a 32-fold induction in fish from the highest 1,1-DCP treatment (16.60 mg/L, MF = 96.3 x 10(-5). Mutations recovered from treated fish showed a distinctive mutational spectrum comprised predominantly of +1 frameshift mutations, induced 166-fold above that of untreated animals. The majority of frameshifts were +1 insertions at thiamine and adenine. These results represent the first evidence of mutagenicity of 1,1-DCP in vivo, and of the highly characteristic spectrum of induced mutations dominated by +1 frameshift mutations. Based upon results from previous in vitro studies, the similar role of glutathione S-transferase (GSTT1-1) in the activation of 1,1-DCP to a mutagen in vivo is also suggested. This study further illustrates the utility of the lamda transgenic medaka as a model for identifying and characterizing potential genetic health risks associated with chemical exposures in the environment.     

Transposition of the Drosophila hydei Minos transposon in the mouse germ line

We tested the suitability of the fly transposon Minos, a member of the Tc1/mariner superfamily, for insertional mutagenesis in the mouse germ line. We generated a transgenic mouse line expressing Minos transposase in growing oocytes and another carrying a tandem array of nonautonomous transposons. The frequency of transposition in the progeny derived from oocytes carrying both transgenes is 8.2%. Analysis of the new integration sites shows a high frequency of transpositions to a different chromosome. Thus Minos transposition could be an effective system for insertional mutagenesis and functional genomic analysis in the mouse.     

On key lesions and all that: a tribute to Paul Lohman

This paper is a tribute to Paul Lohman at the occasion of his retirement from the position of Professor in the Medical Faculty at the Leiden University in The Netherlands and as Director of its Department of Radiation Genetics and Chemical Mutagenesis. Paul's contributions to the science of genetic toxicology are discussed in the context of more recent insights as to how mammalian cells process DNA damage, and how this may lead to cancer and, possibly, aging. Starting with his work on the characterization of UV-induced DNA repair in cultured cells from xeroderma pigmentosum patients and the development of methodology for monitoring the removal of UV-induced lesions in human cells, the concept of the key lesion is introduced. Among the myriad of DNA lesions that can be induced in DNA as a consequence of exposure to a range of natural or synthetic mutagens, key lesions are the ones responsible for subsequent adverse effects, for example, because they give rise to mutation. The development of methods using immunofluorescence microscopy to detect and identify such key lesions and quantitate them at the single cell level, is one of the highlights of Paul's career. Based on the perceived need to evaluate mutational end points in vivo in relation to specific lesions identified by his immunofluorescence methods, Paul subsequently made crucial contributions to the development of the first transgenic mouse model to measure mutations in chromosomally integrated reporter genes. In parallel to his experimental work, Paul greatly contributed to genetic toxicology at the theoretical level by his work on the development and evaluation of methods for assessment or prediction of risks of exposure to environmental mutagens. Finally, Paul has served the discipline of genetic toxicology in a more administrative role in various ways, both locally as one of the founders of the Medical Genetics Center South-West Netherlands and internationally by playing a prominent role in organizations such as ICPEMC. Here, his numerous contributions to the journal Mutation Research, both as author on many papers and as Executive Managing Editor should not go unmentioned.     

Green fluorescent protein (GFP) transgenic fish and their applications

The coupling of the GFP reporter system with the optical clarity of embryogenesis in model fish such as zebrafish and medaka is beginning to change the picture of transgenic fish study. Since the advent of first GFP transgenic fish in 1995, GFP transgenic fish technology have been quickly employed in many areas such as analyses of gene expression patterns and tissue/organ development, dissection of promoters/enhancers, cell lineage and axonal pathfinding, cellular localization of protein products, chimeric embryo and nuclear transplantation, cell sorting, etc. The GFP transgenic fish also have the potentials in analysis of upstream regulatory factors, mutagenesis screening and characterization, and promoter/enhancer trap. Our own studies indicate that GFP transgenic fish may become a new source of novel variety of ornamental fish. Efforts are also being made in our laboratory to turn GFP transgenic fish into biomonitoring organisms for surveillance of environmental pollution.     

High incidence of lung, bone, and lymphoid tumors in transgenic mice overexpressing mutant alleles of the p53 oncogene.

We have investigated the role of the p53 gene in oncogenesis in vivo by generating transgenic mice carrying murine p53 genomic fragments isolated from a mouse Friend erythroleukemia cell line or BALB/c mouse liver DNA. Elevated levels of p53 mRNA were detected in several tissues of two transgenic lines tested. Increased levels of p53 protein were also detected in most of the tissues analyzed by Western blotting (immunoblotting). Because both transgenes encoded p53 proteins that were antigenically distinct from wild-type p53, it was possible to demonstrate that overexpression of the p53 protein was mostly, if not entirely, due to the expression of the transgenes. Neoplasms developed in 20% of the transgenic mice, with a high incidence of lung adenocarcinomas, osteosarcomas, and lymphomas. Tissues such as ovaries that expressed the transgene at high levels were not at higher risk of malignant transformation than tissues expressing p53 protein at much lower levels. The long latent period and low penetrance suggest that overexpression of p53 alone is not sufficient to induce malignancies and that additional events are required. These observations provide direct evidence that mutant alleles of the p53 oncogene have oncogenic potential in vivo and that different cell types show intrinsic differences in susceptibility to malignant transformation by p53. Since recent data suggest that p53 may be a recessive oncogene, it is possible that the elevated tumor incidence results from functional inactivation of endogenous p53 by overexpression of the mutant transgene. The high incidence of lung and bone tumors suggests that p53 transgenic mice may provide a useful model to investigate the molecular events that underlie these malignancies in humans.     

Size Matters: Use of YACs,BACs and PACs in Transgenic Animals

In 1993, several groups, working independently, reported the successful generation of transgenic mice with yeast artificial chromosomes (YACs) using standard techniques. The transfer of these large fragments of cloned genomic DNA correlated with optimal expression levels of the transgenes, irrespective of their location in the host genome. Thereafter, other groups confirmed the advantages of YAC transgenesis and position-independent and copy number-dependent transgene expression were demonstrated in most cases. The transfer of YACs to the germ line of mice has become popular in many transgenic facilities to guarantee faithful expression of transgenes. This technique was rapidly exported to livestock and soon transgenic rabbits, pigs and other mammals were produced with YACs. Transgenic animals were also produced with bacterial or P1-derived artificial chromosomes (BACs/PACs) with similar success. The use of YACs, BACs and PACs in transgenesis has allowed the discovery of new genes by complementation of mutations, the identification of key regulatory sequences within genomic loci that are crucial for the proper expression of genes and the design of improved animal models of human genetic diseases. Transgenesis with artificial chromosomes has proven useful in a variety of biological, medical and biotechnological applications and is considered a major breakthrough in the generation of transgenic animals. In this report, we will review the recent history of YAC/BAC/PAC-transgenic animals indicating their benefits and the potential problems associated with them. In this new era of genomics, the generation and analysis of transgenic animals carrying artificial chromosome-type transgenes will be fundamental to functionally identify and understand the role of new genes, included within large pieces of genomes, by direct complementation of mutations or by observation of their phenotypic consequences.     

Mosquito transgenesis: what is the fitness cost?

The generation of transgenic mosquitoes with a minimal fitness load is a prerequisite for the success of strategies for controlling mosquito-borne diseases using transgenic insects. It is important to assemble as much information as possible on this subject because realistic estimates of transgene fitness costs are essential for modeling and planning release strategies. Transgenic mosquitoes must have minimal fitness costs, because such costs would reduce the effectiveness of the genetic drive mechanisms that are used to introduce the transgenes into field mosquito populations. Several factors affect fitness of transgenic mosquitoes, including the potential negative effect of transgene products and insertional mutagenesis. Studies to assess fitness of transgenic mosquitoes in the field (as opposed to the laboratory) are still needed.     

Reporter genes in transgenic mice

Althoughin vivo models utilizing endogenous reporter genes have been exploited for many years, the use of reporter transgenes to dissect biological issues in transgenic animals has been a relatively recent development. These transgenes are often, but not always, of prokaryotic origin and encode products not normally associated with eukaryotic cells and tissues. Some encode enzymes whose activities are detected in cell and tissue homogenates, whereas others encode products that can be detectedin situ at the single cell level. Reporter genes have been used to identify regulatory elements that are important for tissue-specific gene expression or for development; they have been used to producein vivo models of cancer; they have been employed for the study ofin vivo mutagenesis; and they have been used as a tool in lineage analysis and for marking cells in transplanation experiments. The most commonly usedin situ reporter gene islacZ, which encodes a bacterial -galactosidase, a sensitive histochemical marker. Although it has been used with striking success in cultured cells and in transgenic mouse embryos, its postnatalin vivo expression has been unreliable and disappointing. Nevertheless, the ability to express reporter genes in transgenic mice has been an invaluable resource, providing insights intoin vivo biological mechanisms. The development of newin vivo models, such as those in which expression of transgenes can be activated or repressed, should produce transgenic animal systems that extend our capacity to address heretofore unresolved biological questions.     

Current and future needs for developmental toxicity testing

A review is presented of the use of developmental toxicity testing in the United States and international regulatory assessment of human health risks associated with exposures to pharmaceuticals (human and veterinary), chemicals (agricultural, industrial, and environmental), food additives, cosmetics, and consumer products. Developmental toxicology data are used for prioritization and screening of pharmaceuticals and chemicals, for evaluating and labeling of pharmaceuticals, and for characterizing hazards and risk of exposures to industrial and environmental chemicals. The in vivo study designs utilized in hazard characterization and dose-response assessment for developmental outcomes have not changed substantially over the past 30 years and have served the process well. Now there are opportunities to incorporate new technologies and approaches to testing into the existing assessment paradigm, or to apply innovative approaches to various aspects of risk assessment. Developmental toxicology testing can be enhanced by the refinement or replacement of traditional in vivo protocols, including through the use of in vitro assays, studies conducted in alternative nonmammalian species, the application of new technologies, and the use of in silico models. Potential benefits to the current regulatory process include the ability to screen large numbers of chemicals quickly, with the commitment of fewer resources than traditional toxicology studies, and to refine the risk assessment process through an enhanced understanding of the mechanisms of developmental toxicity and their relevance to potential human risk. As the testing paradigm evolves, the ability to use developmental toxicology data to meet diverse critical regulatory needs must be retained.     

Use of in vivo genetic toxicity data for risk assessment

Mutagenicity studies have been used to identify specific agents as potential carconogens or other human health hazards; however, they have been used minimally for risk assessment or in determining permissible levels of human exposure. The poor predictive value of in vitro mutagenesis tests for carcinogenic activity and a lack of mechanistic understanding of the roles of mutagens in the induction of specific cancers have made these tests unattractive for the purpose of risk assessment. However, the limited resources available for carcinogen testing and large number of chemicals which need to be evaluated necessitate the incorporation of more efficient methods into the evaluation process. In vivo genetic toxicity testing can be recommended for this purpose because in vivo assays incorporate the metabolic activation pathways that are relevant to humans. We propose the use of a multiple end-point in vivo comprehensive testing protocol (CTP) using rodents. Studies using sub-acute exposure to low levels of test agents by routes consistent with human exposure can be a useful adjunct to methods currently used to provide data for risk assessment. Evaluations can include metabolic and pharmacokinetic endpoints, in addition to genetic toxicity studies, in order to provide a comprehensive examination of the mechanism of toxicity of the agent. A parallelogram approach can be used to estimate effects in non-accessible human tissues by using data from accessible human tissues and analogous tissues in animals. A categorical risk assessment procedure can be used which would consider, in order of priority, genetic damage in man, genetic damage in animals that is highly relevant to disease outcome (mutation, chromosome damage), and data from animals that is of less certain relevance to disease. Action levels of environmental exposure would be determined based on the lowest observed effect levels or the highest observed no effect levels, using sub-acute low level exposure studies in rodents. As an example, the known genotoxic effects of benzene exposure at low levels in man and animals are discussed. The lowest observed genotoxic effects were observed at about 1–10 parts per million for man and 0.04–0.1 parts per million in subacute animal studies. If genetic toxicity is to achieve a prominent role in evaluating carcinogens and characterizing germ-cell mutagens, minimal testing requirements must be established to ascertain the risk associated with environmental mutagen exposure. The use of the in vivo approach described here should provide the information needed to meet this goal. In addition, it should allow truly epigenetic or non-genotoxic carcinogens to be distinguished from the genotoxic carcinogens that are not detected by in vitro methods.     

鱼类细胞系在病毒学研究中的应用进展

鱼类细胞培养尽管起步较晚,但截至目前已有近280余株不同的鱼类细胞系相继建立起来,在生理学、病毒学、毒理学、肿瘤及基因工程等多个领域发挥重要作用。主要对鱼类细胞系在病毒学研究中的最新应用,并结合作者自身的研究,尤其侧重于鱼类病毒分离、重要功能基因鉴定、抗病毒免疫和病毒致病机理研究等方面的进展作一概述。     

Transgenic fish

Transgenic fish are produced by the artificial transfer of rearranged genes into newly fertilized eggs. Currently microinjection is the preferred method, although the integration rates of transgenes are generally low. A number of fusion genes, containing retrovirus sequences which direct integration, have been developed to enhance integration of transgenes. Mass gene transfer methods are also being developed. These include lipofection, particle bombardment, and electroporation of embryos and sperm cells. These methods are potentially useful for marine organisms such as crustaceans and molluscs as well as fish. In contrast to microinjection, which treats single cells individually, these methods can transfer genes into a large number of eggs at once. There is some evidence to indicate successful integration and expression of transgenes transferred by the electroporation of embryos and sperm cells. Germline transmission of transgenes has been observed through mating studies, and in some cases the progeny express the new phenotype consistently. However, germline transmission does not necessarily confirm stable integration of the transgene. There is evidence that transgenes may exist extrachromosomally. Transgenic fish are viewed as a useful model for the study of complex biological phenomena such as growth and differentiation, and as a fast track to the production of broodstock for the aquaculture industry. Current research focuses on the elucidation of the mechanisms controlling the regulation of gene expression. The use of transgenic fish for the isolation of developmental genes has just begun. Applications of transgenesis to broodstock development have been focused on the development of fish with accelerated growth, tolerance to low temperature, and disease resistance. However, before the release of transgenic fish into the environment, the possible impact on the environment must be assessed. There must be safeguards to protect the genetic diversities of the natural populations, and to conserve the natural habitats