WO2002034040A2

WO2002034040A2 - Method for generating transgenic fish embryos using an episomal vector system

Info

Publication number: WO2002034040A2
Application number: PCT/EP2001/012246
Authority: WO
Inventors: Alexander D. Crawford
Original assignee: Mermaid Pharmaceuticals Gmbh
Priority date: 2000-10-23
Filing date: 2001-10-23
Publication date: 2002-05-02
Also published as: WO2002034040A3; AU2002221731A1

Abstract

The present invention relates to a novel method for generating transgenic fish embryos using an episomal vector system. In particular, the invention relates to the expression of transgenes in fish embryos or larvae using plasmids containing episomal replication elements derived from the Epstein-Barr episomal vector system.

Description

Method for generating transgenic fish embryos using an episomal vector system

Background of the invention

The analysis of gene function through transgenic methods in model organisms is an accepted approach in functional genomics. Significant progress has been achieved last twenty years in the development of methods for the introduction of exogenous genetic material into the genomes of various model organisms, including rats, mice, frogs, fish, fruitflies, and nematodes. Most of these methods are dependent on the creation of stable transgenic lines, wherein only the progeny of the original founder animals are utilized for the analysis of the physiological effects of the transgene.

For many applications, such as the detailed functional analysis of only small numbers of genes, the generation of stable transgenic lines is an acceptable requirement, especially if the efficiency of germ-line transmission by the original founder animals is reasonably high. Nevertheless, for certain applications, such as the high-throughput analysis of gene function, it would be advantageous to be able to directly analyze the founder generation for the effects of the transgene, especially if the efficiency of germ-line transmission is low. However, such an approach would require the transgenesis method to be able to generate uniformly transgenic animals, as any mosaic expression of the transgene would complicate the phenotypic analysis.

One model organism where this type of approach would be of particular interest is the zebrafish, in part because of the relatively low rate of germ-line transmission of transgenes, but also because of the ease with which transgenic founder animals can be generated, coupled with the attractiveness of the zebrafish as an animal model for human disease (Nasevicius and Ekker, 2000; BarbazUk et al., 2000; Barut and Zon, 2000; Dodd et al., 2000; Long et al., 2000; Wang et al., 1998; Meng et al., 1999a).

Recent work has shown that the most reliable method for the generation of transgenic zebrafish with correct expression of transgenes is one based on the use of large fragments of 5^' regulatory sequences, often in the context of bacterial artificial chromosomes (BACs) or similar vectors, to drive the expression of the transgene (Lin, 2000; Long et al., 1997; Jessen 1998; ibid, 1999; Meng et al., 1999b). Such constructs are injected into zebrafish embryos at the single-cell stage, and the resulting founder animals are raised to adulthood and crossed with wildtype zebrafish to generate F1 progeny that are heterozygous for the transgene.

For this method, usually less than ten percent of the founders are capable of transmitting the transgene to their offspring, and there is strong variation between those founders capable of germ-line transmission with respect to the percentage of their offspring that actually carry the transgene. Both effects are due to the mosaicism of transgene integration and expression, which is due to a combination of factors including the rapid rate of cell division in the early zebrafish embryo, the lack of an easily identifiable pronucleus in the single-cell embryo (in contrast to mouse embryos at the same stage), and the resulting tendency of most injected DNA to be lost from the nuclear compartment into the cytoplasm prior to integration.

It is therefore of great interest to be able to quickly generate transgenic zebrafish with uniform expression of the transgene already within the first generation of injected founders. Such an approach would eliminate the need to raise and breed the founders in order to be able to phenotypically analyse their offspring, and would enable the rapid analysis of larger numbers of different transgenes, as the generation of transgenic zebrafish embryos through microinjection at the single-cell stage is a relatively expedious procedure.

In order to carry out this approach, it is necessary that the method for transgenesis be able to generate embryos wherein all cells within these embryos have a similar number of copies of the transgene, and that the transgene is reliably expressed in all cells. Therefore the method must be independent of integration of the transgene, as this is a stochastic process that occurs on average only after several cell divisions, and it must be independent of position-specific effects, as this would lead to different levels of transgene expression in different cells.

In summary, the ability to generate transgenic fish embryos or larvae within a single generation, having uniform distribution of transgenes throughout most or all cells and tissues, would enable the rapid, cost-effective analysis of gene function for purposes of identifying and validating disease-relevant drug targets as well as for other research applications in drug discovery and genetic engineering. However, until now the generation of transgenic fish has been limited to methods requiring the production of transgenic lines, with stable integrations of transgenes, over at least two generations, thereby precluding the application of this approach for many high throughput applications as described above.

It is thus an object of the present invention to facilitate the generation of transgenic fish embryos or larvae within a single generation, having uniform distribution of transgenes throughout most or all cells and tissues. This object is solved by the provision of a method for establishing uniform distribution of expression vectors in developing fish embryos through the use of episomal vectors delivered to said embryos in early developmental stages.

Description of the invention

Although the invention is described with respect to particular materials and methods or equipment, it is not limited thereto as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Although any materials and methods, or equipment similar or equivalent to those described herein can be used to practice or test the present invention, the preferred equipment, materials and methods are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

The present invention is directed to a method for generating transgenic fish embryos, comprising the introduction of an episomal vector system into fish embryos at the single-cell stage, wherein the episomal vector system contains at least one or more episomal replication elements, a nuclear retention protein, a transgene of interest and a genomic DNA fragment.

In a preferred embodiment of the invention, the episomal replication element is the EBV (Epstein Barr Virus) origin of replication P (oriP) or a portion or a derivative thereof (Yates et al., 1985; Margolskee et al., 1988; Young et al., 1988; Calos, 1998). The EBV oriP can be replaced by episomal replication elements from other viruses referred to as derivatives of EBV oriP. Therefore, another embodiment of the invention uses the BPV (Bovine Papilloma Virus) origin of replication or a portion or a derivative thereof as an episomal replication element (Ashman et al., 1985; Gilbert et al., 1987; Ravnan et al., 1992).

In another preferred embodiment of the invention, the portion of oriP of EBV comprises a family of repeats. The family of repeats acts as a binding site for the EBV protein EBNA-1 (Yates, 1984; Reisman et al., 1985; Lupton et al., 1985; Wang et al., 1997).

The EBV oriP contains multiple sequence elements with different functions, including the region of dyad symmetry and the family of repeats. The region of dyad symmetry provides the episomal plasmid with the ability to initiate DNA replication during the S phase of the cell cycle and is known to function in this capacity only in primate and canine cells (Yates et al., 1991). A further component of the episomal vector system according to the present invention is the nuclear retention protein, e.g. the EBV EBNA-1 protein. The family of repeats acts as a binding site for the EBV EBNA-1 protein. The EBNA-1 protein binds to both the EBV ori and to the endogenous chromatin during chromosomal segregation, thereby acting as a nuclear retention mechanism for the plasmid during mitosis (Wendelburg et al., 1998).

A further component of the episomal vector system according to the present invention is a transgene of interest, wherein the term transgene of interest denotes a nucleic acid molecule encoding a protein or untranslated RNA molecule of either known or unknown function. The transgene of interest can be either native or foreign to the transgenic animal generated herewith.

In one embodiment of the invention, the transgene of interest encodes a protein or untranslated RNA molecule of known or unknown function (gene products). Gene products with unknown functions of particular interest include secreted proteins, receptors, ion channels, enzymes, proteases, kinases, and phosphatases, with the aim of elucidating the function of potential therapeutic proteins or defined molecular targets for the identification of pharmacological compounds in drug discovery applications.

In another embodiment of the invention, gene products with known functions of particular interest include those capable of generating disease-like phenotypes when overexpressed, with the aim of generating fish embryos or larvae with medically relevant index phenotypes for modifier screens to identify genes or gene products functioning as enhancers or suppressors of said index phenotypes through various inactivation methods, including chemical compounds.

In one embodiment of the invention, the transgene of interest encodes a marker gene product. The term marker denotes easily identifiable proteins for the detection of particular cells, tissues, for the quantification of activities of particular promoters, and for the measurement or detection of particular physiological events or cellular activities. In a preferred embodiment of the invention, marker gene products are selected from the group consisting of GFP, β-lactamase and lacZ (Amsterdam et al., 1995; ibid, 1996; Chalfie et al., 1994; Raz et al., 1998; Lin et al., 1994).

The episomal vector system of the present invention further comprises a genomic DNA fragment. In a preferred embodiment, the genomic DNA fragment is at least 5 kb long, wherein a length of 6 to 10 kb is particularly preferred. The genomic DNA can be of fish or mammalian origin. Said genomic DNA fragment enables the replication of EBV-based vectors in non-primate and non-canine cells (Simson et al., 1996a; ibid, 1996b; Tolmachova et al., 1999; Wade-Martins et al., 1999).

Until recently, it was thought that EBV vectors could not function in non-primate and non- canine cells, as the function of the dyad symmetry element in the EBV ori was restricted to these species. However, it has now been demonstrated that long fragments of genomic DNA, when integrated into EBV-based vectors, act as DNA replication elements when these vectors are introduced into mammalian cells, including cells from mammals other than primate and canine species (Krysan et al., 1989; ibid, 1991 ; ibid, 1993).

The use of at least several kilobases of DNA, preferably at least 5 kilobases, in particular 6 to 10 kilobases, encoding promoter sequences to direct expression of a transgene of interest is recommended for two reasons: (1) to improve the reliability and fidelity of the promoter with respect to the level and specificity of transgene expression (Huertas et al., 2000), and (2) to provide the vector with the ability to initiate DNA replication independently of the EBV ori, as the latter is only known to function in primate and canine cells.

Nevertheless, there has not yet been evidence to suggest that said phenomenon, namely of episomal plasmid replication enabled through origins of DNA replication in genomic DNA fragments, is also true for non-mammalian species (Caddie et al., 1982; Haase et al., 1991). In addition, it has been unclear whether the function of EBNA-1 as a nuclear retention signal for episomal plasmids is dependent on mammalian-specific co-factors (Wang et al., 1997; Chen et al., 1998; Medina et al., 2000), given that EBV is a primate-specific virus.

The method of the present invention enables episomal plasmid replication in non- mammalian species.

In one embodiment said method provides for plasmids containing EBV elements, including the portion of the origin of replication containing the family of repeats, but not the complete family of repeats, a genomic DNA fragment of at least 5 kb, preferably 6-10 kb in length, together with the necessary elements for expression of a transgene, including promoter elements providing either ubiquitous, tissue-specific, or inducible expression of the transgene, thus enabling the functional analysis of genes with multiple functional roles in vertebrate development.

Several methods are possible to generate a vector system that is capable of replicating in all or most cells of the developing fish embryo. In one embodiment of the invention, the vector contains EBV oriP family of repeats, a transgene of interest, a nuclear retention protein like the EBV protein EBVA-1 and a genomic DNA fragment. In a preferred embodiment the genomic DNA fragment contains a promoter, for example an expression cassette for the transcription of RNA encoding the EBNA-1 protein.

10

The episomal vector system can be introduced into the fish embryos by any methods known to the person skilled in the art, such as microinjection or transfection. If the episomal vector system is introduced by transfection, it is carried out in the presence of DNA carrier agents or membrane-permeabilizing agents, wherein said DNA carrier agents or membrane- 1.5 permeabilizing agents include lipofection agents, liposomes, or polyamine.

In further embodiments of the invention, the episomal vector system is introduced into the fish embryos by electroporation or by ballistic methods (DNA gun).

20 In another embodiment of the invention, the gene encoding the nuclear retention protein, e.g. the EBNA-1 gene, is in a separate expression vector that is either co-injected with the first or that is used to generate stable transgenic lines expressing the necessary levels of EBNA-1 protein.

25 EBV-based episoma] vectors constructed according to this invention are introduced into fish embryos at the single-cell stage. Upon introduction of the episomal vector system, the fish embryos are monitored to assure acceptable levels of transgene expression, which can be done by including a visible reporter such as green fluorescent protein in the vector as the second cistron in a bicistronic expression cassette. For most phenotypic assays the

30 embryos need be maintained only several days, thereby minimizing any long-term complications with respect to plasmid maintainance and stability. In one embodiment of the invention, the episomal vector system is introduced into teleost fish embryos. Particularly preferred teleost embryos are embryos of the species Danio rerio (zebrafish) or Oryza latipes (medaka).

In another embodiment, said fish embryos are homozygous or heterozygous for mutations. In a further preferred embodiment, the fish embryos are transgenic fish embryos.

The method of the present invention can be modified by providing the nuclear retention protein in a separate expression vector.

In one embodiment of the invention, the nucleic acid sequence encoding the nuclear antigen EBNA-1 is cloned into a DNA expression vector, and said DNA expression vector is co-introduced into the fish embryo with the episomal vector system.

In another embodiment, the nucleic acid sequence encoding the nuclear antigen EBNA-1 is cloned into an in vitro RNA expression vector, from which EBNA-1 cRNA is synthesized. Said cRNA is co-introduced into the fish embryo with the episomal vector system (for an example of multiple episomal vectors in mammalian cells see Horlick et al., 2000).

The term in vitro RNA expression vector denotes a vector wherein transcription is driven by a phage RNA promoter such as T7, T3 or SP6.

The mRNA can be introduced into early embryos by microinjection or any other method known to the person skilled in the art.

The invention described herein is further illustrated by the following examples:

Example 1

Generation of an episomal vector for expression of a transgene of interest (GFP) in zebrafish embryos

A 750bp DNA fragment containing an engineered EF1-alpha promoter/enhancer derived from Xenopus laevis is restricted from the plasmid pXeX (Johnson and Krieg, 1994), and ligated into the sites of the MCS of pDsRed2-1 (Clontech, Inc.) to create pDsRed2-1-XeX (abbreviated pXR). A 1500bp DNA fragment containing the family of repeats region (FRR) of the EBV oriP is amplified from pREP4 (Invitrogen, Inc.) using long-range PCR and high- fidelity thermostable DNA polymerase (Advantage HF-PCR system, Clontech, Inc.) according to the manufacturer's specifications, and ligated into pXR to create pXR-FRR (abbreviated pXRF). High-molecular weight genomic DNA is isolated from zebrafish larvae using genomic DNA purification columns (Qiagen GmbH) according to the manufacturer's specifications, restricted with Hindlll, and ligated into pXRF to create pXRF-gDNA (abbreviated pXRFD). Individual pXRFD clones are isolated, characterized through restriction analysis to determine the size of genomic DNA inserts, bacterially amplified and purified using plasmid DNA purification columns (Qiagen) according to the manufacturers specifications. An IδOObp DNA fragment containing the EBNA-1 gene is amplified from pREP4 (Invitrogen, Inc.) using long-range PCR and high-fidelity thermostable DNA polymerase (Advantage HF-PCR system, Clontech, Inc.) according to the manufacturer's specifications, and ligated into pBluescript to create pBEBNA- Following linearization, cRNA synthesized from pBEBNA-1 (Message Machine system, Ambion, Inc. according to the manufacturer's protocol) is injected into 1-cell stage zebrafish embryos at a concentration of 50 ng/microliter together with purified pXRFD plasmid (as described in Westerfield, 1993).

The distribution of the RNA is tested at different stages of development using EBNA-1 as a hybridization probe in whole-mount in situ hybridizations (as described in Westerfield, 1993). Maintainance and distribution of pXRFD in developing larvae is determined through visualization of DsRed fluorescence (as described in Amsterdam et al., 1996 using specifications for DsRed visualization as provided by Clontech, Inc. under www.clontech.com).

Example 2

Generation of zebrafish embryos with tissue-specific expression of a transgene of interest (GFP) A DNA fragment containing the zebrafish insulin promoter isolated from zebrafish genomic DNA according to established methods and ligated into the sites of the MCS of pDsRed2-1 (Clontech, Inc.) to create pDsRed2-1-in (abbreviated pinR), A 1500bp DNA fragment containing the family of repeats region (FRR) of the EBV oriP is amplified from pREP4 (Invitrogen, Inc.) using long-range PCR and high-fidelity thermostable DNA polymerase polymerase (Advantage HF-PCR system, Clontech, Inc.) according to the manufacturer's specifications, and ligated into pinR to create pinR-FRR (abbreviated pinRF). High- molecular weight genomic DNA is isolated from zebrafish larvae using genomic DNA purification columns (Qiagen GmbH) according to the manufacturer's specifications, restricted with Hindlll, and ligated into pinRF to create pinRF-gDNA (abbreviated pinRFD). Individual pinRFD clones are isolated, characterized through restriction analysis to determine the size of genomic DNA inserts, bacterially amplified and purified using plasmid DNA purification columns (Qiagen) according to the manufacturers specifications. An 1800bp DNA fragment containing the EBNA-1 gene is amplified from pREP4 (Invitrogen, Inc.) using long-range PCR and high-fidelity thermostable DNA polymerase (Advantage HF- PCR system, Clontech, Inc.) according to the manufacturer's specifications, and ligated into pBluescript to create pBEBNA-1. Following linearization, cRNA synthesized from pBEBNA-1 (Message Machine system, Ambion, Inc. according to the manufacturer's protocol) is injected into 1-cell stage zebrafish embryos at a concentration of 50 ng/microliter together with purified pinRFD plasmid (as described in Westerfield, 1993).

The distribution of the RNA is tested at different stages of development using EBNA-1 as a hybridization probe in whole-mount in situ hybridizations (as described in Westerfield, 1993). Maintainance and distribution of pinRFD in developing larvae, as well as tissue- specific expression of DsRed from pinRFD, is determined through visualization of DsRed fluorescence (as described in Amsterdam et al., 1996 using specifications for DsRed visualization as provided by Clontech, Inc. under www.clontech.com).

Example 3 Generation of zebrafish embryos with tissue-specific expression of a transgene of interest (an endogenous gene) together with a marker gene (GFP)

The strategy of example 2 is used, except that pinR, prior to modification with the family of repeats region (FRR) to create pinRF, is modified through ligation of a cDNA of interest and an internal ribosomal entry sequence (IRES) into the MCS between the insulin promoter and the DsRed gene to create pinR-cDNA-lRES (abbreviated pinCIR). A 1500bp DNA fragment containing the family of repeats region (FRR) of the EBV oriP is amplified from pREP4 (Invitrogen, Inc.) using long-range PCR and high-fidelity thermostable DNA polymerase (Advantage HF-PCR system, Clontech, Inc.) according to the manufacturer's specifications, and ligated into pinCIR to create pinCIR-FRR (abbreviated pinCIRF). High- molecular weight genomic DNA is isolated from zebrafish larvae using genomic DNA purification columns (Qiagen GmbH) according to the manufacturer's specifications, restricted with Hindlll, and ligated into pinCIRF to create pinCIRF-gDNA (abbreviated pinCIRFD). Individual pinCIRFD clones are isolated, characterized through restriction analysis to determine the size of genomic DNA inserts, bacterially amplified and purified using plasmid DNA purification columns (Qiagen) according to the manufacturers specifications. An 1800bp DNA fragment containing the EBNA-1 gene is amplified from pREP4 (Invitrogen, Inc.) using long-range PCR and high-fidelity thermostable DNA polymerase (Advantage HF-PCR system, Clontech, Inc.) according to the manufacturer's specifications, and ligated into pBluescript to create pBEBNA-1. Following linearization, cRNA synthesized from pBEBNA-1 (Message Machine system, Ambion, Inc. according to the manufacturer's protocol) is injected into 1-cell stage zebrafish embryos at a concentration of 50 ng/microliter together with purified pinCIRFD plasmid (as described in Westerfield, 1993).

The distribution of the RNA is tested at different stages of development using EBNA-1 as a hybridization probe in whole-mount in situ hybridizations (as described in Westerfield, 1993). Maintainance and distribution of pinCIRFD in developing larvae, as well as tissue- specific expression of DsRed from pinCIRFD, is determined through visualization of DsRed fluorescence (as described in Amsterdam et al., 1996 using specifications for DsRed visualization as provided by Clontech, inc. under www.clontech.com). Effects of the expression of the cDNA are determined by phenotypic analysis, including morphological, histochemical, immunohistochemical, and molecular assays.

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Claims

1. A method for generating transgenic fish embryos, comprising the introduction of an episomal vector system into fish embryos at the single-cell stage, wherein the episomal vector system contains at least one or more episomal replication elements, a nuclear retention protein, a transgene of interest and a genomic DNA fragment.

2. A method according to claim 1 , wherein the episomal replication element is the EBV (Epstein Barr Virus) origin of replication P (oriP) or a portion or a derivative thereof.

3. A method according to claim 2, wherein the portion of oriP comprises a family of repeats.

4. A method according to claim 1, wherein the episomal replication element is the BPV (Bovine Papilloma Virus) origin of replication or a portion or a derivative thereof.

5. A method according to anyone of claims 1 to 4, wherein the nuclear retention protein is the EBV protein EBNA-1.

6. A method according to any one of claims 1 to 5, wherein the transgene of interest encodes a protein or untranslated RNA molecule of known or unknown function.

7. A method according to claim 6, wherein the protein or untranslated RNA molecule of unknown function is selected from the group consisting of secreted proteins, receptors, ion channels, enzymes, proteases, kinases and phosphatases.

8. A method according to claim 6, wherein the protein or untranslated RNA molecule of known function is selected from the group consisting of proteins capable of generating disease-like phenotypes when overexpressed.

9. A method according to claim 6, wherein the transgene of interest encodes a marker gene product.

10. A method according to claim 9, wherein the marker gene product is selected from the group consisting of GFP, β-lactamase and lacZ.

11. A method according to claims 1 to 10, wherein the promoter provides ubiquitous, tissue-specific or inducible expression of the transgene of interest.

12. A method according to any one of claims 1 to 11 , wherein the genomic DNA fragment is at least 5 kb long.

13. A method according to claim 1 , wherein introduction of the episomal vector system into the fish embryo is carried out by microinjection.

14. A method according to claim 1, wherein introduction of the episomal vector system into the fish embryo is carried out by transfection.

15. A method according to claim 14, wherein transfection is carried out in the presence of DNA carrier agents or membrane-permeabilizing agents.

16. A method according to claim 15, wherein said DNA carrier agents or membrane- permeabilizing agents include lipofection agents, liposomes, or polyamine.

17. A method according to claim 1, wherein introduction of the episomal vector system into the fish embryo is carried out by electroporation.

18. A method according to claim 1, wherein introduction of the episomal vector system into the fish embryo is carried out by ballistic methods (DNA gun).

19. A method according to any one of claims 1 to 18, wherein said fish embryos are teleost embryos.

20. A method according to claim 19, wherein said teleost embryos are embryos of the species Danio rerio (zebrafish) or Oryza latipes (medaka).

21. A method according to any one of claims 1 to 20, wherein said fish embryos are homozygous or heterozygous for mutations.

22. A method according to any one of claims 1 to 21 , wherein said fish embryos are transgenic.

23. A method for generating transgenic fish embryos, comprising the introduction of an episomal vector system into fish embryos at the single-cell stage, wherein the episomal vector system contains at least one or more episomal replication elements, a nuclear retention protein, a transgene of interest and a genomic DNA fragment, wherein the nuclear retention protein is contained within a separate expression vector.

24. A method according to claim 23, wherein the separate expression vector containing the nuclear retention protein is a DNA or in vitro RNA expression vector.

25. A method according to claim 24, herein the in vitro RNA expression vector is used to synthesize cRNA of the nuclear retention protein.

26. A method according to claims 23 and 24, wherein the DNA expression vector containing the nuclear retention protein and the episomal vector system are co-introduced into fish embryos.

27. A method according to claim 25, wherein the cRNA of the nuclear retention protein synthesized from the in vitro RNA expression vector and the episomal vector system are co- introduced into fish embryos.

28. A method according to claims 23 to 27, wherein the nuclear retention protein is the EBV protein EBNA-1.