Field of the Invention

This invention relates to the expression of nucleic acid sequences, typically heterologous sequences, encoding pharmaceutically or biologically active proteins, in fish. This generates recombinant polypeptides which can then be harvested from the fish or their eggs.

Background to the Invention Certain systems exist for the expression of heterologous recombinant proteins.

These include microbial systems, such as yeast and E. coli, and animal systems of two types, tissue culture and whole animal systems.

Microbial systems suffer from the drawback that they often fail to glycosylate or fold the heterologous protein correctly. In many cases, this means that the protein is biologically inactive, especially when an eukaryotic protein is produced in a prokaryotic system like E. coli where no glycosylation occurs.

Cell cultures, typically mammalian cell cultures, are an alternative as they are capable of effecting folding and glycosylation. However, while cell culture involves relatively simple experimental procedures and produces rapid reproducible results, it is extremely expensive; using recombinant Factor VIII prepared Chinese Hamster

Ovary (CHO) cells, it costs around £80,000 per year to treat a haemophilia patient.

A further alternative is the introduction of cloned sequences into whole animal systems in vivo, which should maximise the possibility of correct expression of the gene construct since the introduced sequences are exposed to a complete spectrum of cell-specific signals. For example, a less expensive way to produce heterologous proteins than using cell cultures is to produce the protein in the milk of transgenic cows or sheep. Serious problems remain, however. The lytic enzymes contained in the milk hydrolyse the heterologous proteins unless they are highly resistant to degradation. Further, large mammals such as cows and sheep require extensive care. Also, as some diseases are common to non-human mammals and humans, there may be a danger that recombinant products produced in mammals will transmit pathogenic viruses and/or prions to human patients.

Recently, fish have been used to express heterologous proteins and a number of workers have produced stable transgenic fish lines which express a transgene in the Fl generation: these include lines expressing genes which encode, amongst others, growth hormone and antifreeze protein, as well as bacterial reporter genes such as CAT and LacZ.

Fish have a number of advantages for expression of recombinant proteins: (i) they are cheap to produce in large numbers; (ii) technology is available for the production of large numbers of transgenic fish; and (iii) as no known viruses or prions infect both humans and fish, transmission of disease is highly unlikely.

Summary of the Invention

The present inventors have found that, using a construct comprising DNA encoding the human Factor VII protein under the control of the CMV promoter, it is possible to express Factor VII transiently in transgenic zebrafish embryos. Furthermore, the recombinant Factor VII protein produced in these embryos is biologically active. Thus, it has been demonstrated that transgenic fish can be used to produce biologically active pharmaceutical polypeptides, especially in their embryonic tissues.

The inventors have also characterised the 5' region of the gene for a new member of the carp myosin heavy chain (MyoHC) gene family, the FG2 myosin heavy chain gene (Gauvry et al. (1996) Eur. J. Biochem, Vol 236; 887-894, incorporated herein by reference). Sequencing of the 5' region has revealed the presence of TATA and CCAAAT boxes typical of a promoter region. These motifs are located 30 bp and 74 bp, respectively, upstream of the transcription start site. The sequence also revealed several E box (CANNTG) motifs between positions -896 and

-629 relative to the transcription start site. Furthermore, a putative MEF2 binding site (GCTATATTTA) is located at position -860. When this upstream promoter region was attached to a reporter gene, it was found to drive tissue-specific expression in the skeletal muscle of carp {Cyprinus carpio) embryos as well as in the muscle of mature fish.

Accordingly the present invention provides an expression construct for expressing a polypeptide in fish, which construct comprises a nucleic acid coding

sequence encoding the polypeptide operably linked to a regulatory sequence capable of directing the expression of the coding sequence in a cell of the fish

Preferably, the regulatory sequence is capable of directing expression of the coding sequence in, for example, a cell of a fish egg, a cell of an embryonic tissue, a cell of an extra-embryonic tissue, for example, in a cell of the yolk syncytial layer

(YSL) and/or enveloping layer (EVL) of the fish egg In another embodiment, the regulatory sequence is capable of directing expression of the coding sequence in a cell of the skeletal muscle

Preferably, the polypeptides are pharmaceutical products with biological activity These may be human factor VII, human factor VIII, human factor IX, calcitonm, an mterleukm, an interferon, erythropoietin, tumour necrosis factor, α- galactosidase, adenosine deammase, or any functionally active part of such a protein

The present invention also provides a nucleic acid vector which comprises an expression construct of the invention The invention further provides a method for producing a transgenic fish, zygote, egg or embryo which method composes introducing episomally an expression construct of the invention into a cell of the fish, zygote, egg or embryo In another embodiment, the method of the invention compnses chromosomally incorporating a construct of the invention into the genome of the fish, zygote, egg or embryo The fish species involved will be of taxa Cyprimdae, Cichhdae, Salmonidae,

Clandae, S umidae or Ictaluridae, and will include zebrafish, {Danw rerio), African catfish {Clarias ganepinus), rainbow trout {Orcorhynchus my ss), carp {Cyprinus carpi ), or tilapia {Oreochromis nύoticus)

The invention also provides a method for of producing a recombinant polypeptide, which method composes expressing the polypeptide encoded by the coding sequence of the construct of the invention in a fish, fish egg or embryo and recoveπng the polypeptide

Preferably, the expression construct is introduced by microinjection Alternatively it may be introduced by electroporation or hpofection Preferably the regulatory sequences compnse promoters giving ubiquitous or tissue specific expression, and include such regulatory sequences, mtrons, enhancers, polyA elements and other nucleic acid sequences as are necessary to maximise

expression in some or all tissues of fish eggs, embryos or adults. The regulatory sequences, in particular promoter sequences, used in the expression constructs are of viral or animal origin, are capable of driving expression in fish cells.

The expression of the coding sequences will be either transient, in which case the recovery will be from eggs or embryos, or stable, in which case the recovery will be from embryos or young fish, or mature fish.

Expression of polypeptides may be achieved by introducing the expression construct of the invention directly into somatic tissues of the adult fish preferably muscle tissue/cells. The gene constructs may also be introduced into germ line tissue/cells including gametes to achieve transmission to progeny fish.

Fish which have been made transgenic and which express the transgene copy or copies in a stable and chromosomally integrated manner may also be used for breeding so that eggs, embryos and other generations of expressing cells and fish can be produced.

Detailed Description of the Invention

It is possible in fish to express proteins, typically heterologous proteins, having pharmaceutical or biological properties. This technique allows the production of properly glycosylated and folded proteins, thus avoiding the disadvantages of microbial expression systems but is relatively inexpensive compared to the use of mammalian cell cultures or large mammalian hosts which, albeit for different reasons, both require a labour-intensive approach.

In particular, two general approaches are possible. (i) Using a regulatory sequence capable of directing expression in fish muscle, or in another tissue, nucleic acid constructs incorporating the regulatory sequence and a coding sequence for a polypeptide of interest can be introduced into fish at a suitable stage in their development. The protein is then recovered from the muscle, and/or from other tissues, by harvesting the fish. For this approach, muscle- specific promoters, especially the myosin heavy chain (MyoHC) promoter, are preferred.

(ii) Polypeptides of interest can be expressed in and recovered from fish eggs, which eggs may be fertilised or unfertilised; or from developing zygotes or embryos. For example, a transgenic sexually mature fish can be produced which expresses the polypeptide of interest, under the control of a suitable the regulatory sequence, in her eggs. These eggs can then be removed and the protein extracted.

This system has the advantage that no mature transgenic fish need be sacrificed to obtain the recombinant polypeptide. For this approach, a regulatory sequence capable of directing expression in a fish egg or developing zygote or embryo is required.

The polypeptides of the invention are produced by expressing DNA encoding them using an expression construct of the invention which comprises a nucleic acid coding sequence encoding the polypeptide operably linked to a regulatory sequence capable of directing the expression of the coding sequence in a cell of the fish.

The nucleic acid coding sequence may be DNA or RNA, preferably DNA, typically genomic DNA or cDNA. It may encode a polypeptide of any suitable length.

Any suitable polypeptide can be produced according to the invention, by expressing a nucleic acid, i.e. DNA or RNA, encoding it. Typically, such a polypeptide will be heterologous with respect to the fish system, i.e. it will be a polypeptide not naturally produced by the fish. However, it may be a homologous polypeptide.

Typically, the polypeptides produced in this way will have pharmaceutical and/or biological properties. Preferably the polypeptides of the invention are suitable for pharmaceutical use in mammals, more preferably humans. For example, they may be useful in treating, preventing or ameliorating a disease or other pathological condition of a human or animal subject.

Preferred polypeptides that may be produced in this way include blood clotting factors such as factors VII, VIII and IX; calcitonin; interleukins; interferons such as α-, β- and γ- interferon; α-galactosidase; tumour necrosis factor (TNF); and adenosine deaminase. Preferably, such polypeptides will be produced in their complete (native) form, although the nucleic acid sequences of the invention may also encode functionally active parts of these polypeptides, for example parts of such polypeptides that have

the activity, or some of the activity, of the complete polypeptide. Similarly, the nucleic acid sequences of the invention may encode derivatives, for example mutated versions of the native polypeptides. For example, they may encode versions of the polypeptide that differ at one or more amino acids from the native version. Alternatively, the nucleic acid sequences may differ from the native sequence but encode the same amino acid sequence. Thus, the nucleic acid sequences may be degenerate of the native sequences. In this respect, the DNA sequences may be altered in such a way as to include codons that are preferred for expressing particular amino acids in fish cells; i.e. codons that are less efficiently expressed in fish may be replaced by more efficiently expressed ones.

In the constructs of the invention, the regulatory sequence of the invention is operably linked to a regulatory sequence capable of directing its expression in a cell of a fish. "Operably linked" refers to a juxtaposition wherein the regulatory sequence and the nucleic acid sequence encoding the polypeptide of the invention activity are in a relationship permitting the coding sequence to be expressed under the control of the promoter. Thus, there may be elements such as 5' non-coding sequence between the regulatory sequence and coding sequence, as long as they enhance, or do not impair, the correct control of the expression of the coding sequence by the regulatory sequence. A regulatory sequence comprises at least a promoter sequence. In addition, a regulatory sequence may comprise other elements such as some or all of the following: a regulator of the promoter; an enhancer for the promoter; and/or a translational start site. The expression construct may also comprise a transcriptional terminator 3' to the sequence encoding the polypeptide of the invention. The expression construct may also comprise one or more introns or other non-coding sequences, for example, 3' or 5' to the sequence encoding the polypeptide of the invention. Such sequences can be included in the construct if they enhance or do not impair the correct control of the coding sequence by the promoter. The construct may also comprise a polyadenylation (poly A) signal operably linked 3' to the nucleic acid coding sequence, for example the SV40 polyA signal or the Chinook Salmon polyA signal. The constructs of the invention may also optionally comprise one or more Locus Control Regions (LCRs). These act as enhancers for certain fish genes and can

lmprove transgene expression (Aronrow et al (1995) Mol Cell Biol Vol 15, 1123- 1135)

The constructs of the invention may be included within a vector, suitably a rephcable vector, for instance a rephcable expression vector In addition to the expression construct of the invention, a vector of the invention typically composes an oogin of replication so that the vector can be replicated in a host cell such as a bacteoal host cell or a yeast host cell A vector may also comprise additional elements involved in the control of expression of the coding sequence as discussed above The vector may also contain one or more selectable marker genes, typically an antibiotic resistance gene, for example an ampicilhn resistance gene for the identification of bacterial transformants, or a marker gene that allows selection of yeast transformants Optionally, the vector may also compose an enhancer for the promoter The expression vector may be of any type The vector may be in linear or circular form For example, the construct may be incorporated into a plasmid vector or a viral vector Those of skill in the art will be able to prepare suitable vectors and constructs of the invention starting with widely available vectors which will be modified by genetic engmeeong techniques such as those descobed by Sambrook et al (Molecular Cloning A Laboratory Manual, 1989, Coldspong Harbor Press, NY, USA)

Any suitable regulatory sequence may be used in the constructs of the invention, as long as the regulatory sequence is capable of directing expression in a cell of a fish

Regulatory sequences in the expression construct of the invention may be capable of directing expression in cells of some or all of the tissues of a fish For example, they may direct expression wholly or mainly in certain tissues Thus, they may be tissue-specific. For example, they may direct expression wholly or mainly m one or more particular fish tissues Regulatory sequences that are specific for tissues of the fish egg or skeletal muscle are preferred It is preferred that the regulatory sequence directs expression in a cell of a fish egg such as a cell of an embryonic or extra-embryonic tissue of a fish egg For example, the promoter may direct expression in a cell of an extra-embryonic tissue

such as a cell of the yolk syncytial layer (YSL) or enveloping layer (EVL) of the egg.

It is also preferred to use promoters capable of directing expression of the coding sequence in a cell of the muscle, typically the skeletal muscle, of a fish.

Regulatory sequences in the construct of the invention may be constitutive or inducible.

Specifically, one class of preferred regulatory sequences are the myosin heavy chain gene promoters. In particular, the caφ FG2 myosin heavy chain promoter described by Gauvry et al. (1996), ibid, is preferred. This is a muscle-specific promoter, in the sense that it directs expression in cells of the skeletal muscle. Unexpectedly, it also directs expression in the YSL and EVL. A particularly preferred region of the FG2 MyoHC promoter is from the transcription start site to

901 bp upstream. This type of tissue-specific regulatory sequence is particularly preferred when the expression construct is to be stably introduced into the fish genome long-term, especially into the genome of germline cells. More generally, fish-derived tissue-specific regulatory sequences are preferred where long-term expression and/or stable integration of the expression construct into the fish cell genome, rather than transient expression is required. It is also preferred to use promoters which are inducible such that expression of the polypeptide can be induced by a suitable stimulus at the desired time. This may avoid any side-effects associated with the continuous expression of a heterologous polypeptide.

Other preferred regulatory sequences include the cytomegalovirus (CMV) promoters; (e.g. the CMV 1E1 promoter) the Friend murine leukaemia virus long terminal repeat promoter (MuLV-LTR); the thymidine kinase, (TK) promoter, optionally in combination with the Rous Sarcoma Virus (RSV) enhancer; the mouse metallothionein I (rnMTHI) promoter; the Xenopus elongation factor lα (eflα) promoter, optionally in combination with the eflα enhancer; the caφ β-actin promoter; the mouse Hox 1.3 promoter; the rat GAP 43 gene promoter; the SV40 early promoter; the rabbit β-cardiac myosin heavy chain promoter; and the human MxA promoter. Particularly preferred for transient expression are ubiquitous and/or constitutive regulatory sequences allowing high level expression in all cell types.

Viral regulatory sequences, for example CMV and RSV sequences are especially preferred.

Optionally, an enhancer may also be used in addition to the promoter. The enhancer will be chosen to be compatible with the promoter. Some suitable enhancers include the rat foetal light chain enhancer, the eflα enhancer and the RSV enhancer.

Any suitable type of fish may be used, according to the invention, for the production of polypeptides of interest. Teleost fish are preferred. Amongst teleost fish, preferred taxa include Cyprinidae, Cichlidae, Salmonidae, Claridae, Siluridae and Ictaluridae. Some particularly preferred types of fish are caφ (e.g. Cyprinus carpio), zebrafish {Danio rerio), African catfish {Clarias gariepinus), tilapia {Oreochronis niloticus) atlantic salmon {Salmo Salar), rainbow trout {Oncorhyncus mykiss), medaka {Oryzias latipes). Generally, when it is desired to recover polypeptides from fish eggs, or from developing zygotes or embryos preferred fish for use according to the invention are able to produce large eggs and/or large numbers of eggs. Also, it is desirable to use fish for which propagation and/or transgenic technology is well developed. The present invention also provides cells harbouring the vectors or constructs of the invention. These cells may be of any type. For example, they may be microorganism cells. Thus, they may for example be bacterial, e.g. E. coli, or yeast cells.

Preferred cells of the invention are fish cells. Cells of any type of fish are within the scope of the invention, and cells of the fish taxa mentioned herein are preferred. Similarly, the cells may be cells of any type of fish tissue, although tissues mentioned herein are preferred. Cells of the fish egg, for example cells of embryonic tissues, or of extra-embryonic tissues such as the YSL and EVL, are particularly preferred. Muscle cells, typically skeletal muscle cells are also preferred. Fish cells of the invention may be in any form. They may be isolated, e.g. in culture, or in a fish, e.g. a mature fish; or in a fish zygote, embryo or egg. Thus, the invention provides transgenic fish comprising cells of the invention. These may be at any stage of development. Thus, for example, the invention provides mature and immature transgenic fish, as well as transgenic fish zygotes, embryos and eggs. In such fish, some or all of the cells harbour constructs or vectors of the invention. Thus, the fish may be uniformly transgenic or transgenic "mosaics" in which only some cells are transgenic. Cells of the invention may be localised in particular tissue types,

for example in one or more tissues such as the skeletal muscle, germline cells or eggs. The transgenic fish of the invention may be of either sex; i.e. they may be male or female.

The constructs and vectors of the invention may be introduced into cells, to make cells of the invention, by any suitable means (for example, as set out in

Sambrook et al. (1989) ibid). For example, the cells may be transformed or transfected by any means known in the art. For example, constructs and vectors of the invention may be packaged into infectious viral particles, for example retroviral, particles or lambda virus particles which are then used to transfer the constructs or vectors to the cells. Also, they may be introduced by micro injection, electroporation, calcium phosphate precipitation, biolistic methods, (e.g. tungsten bombardment) or by contacting naked nucleic acid vectors or constructs with the cells in solution. Optionally, agents that facilitate DNA transfer into cells may be used. These include liposomes. When DNA or RNA is introduced into fish eggs, a preferred means of delivery is microinjection, using techniques known to a person of skill in the art.

Microinjection can be performed at any suitable stage in the development of the egg, for example prior to fertilisation; or at the one-cell, two-cell, four-cell or eight-cell stage. Another preferred technique for delivering nucleic acids to fish eggs, as well as to fish sperm, is electroporation which is reviewed extensively in Transgenic Animals - Generation and Use (1997) pp 129-132, ed. L. M. Houdebin, Harwood Acedemic

Publishers.

The vectors or constructs introduced into cells may be of any suitable type. For example, they may be able to integrate constructs of the invention into the cell genome or they may be episomal, and remain free in the cell. The invention also provides methods of making transgenic fish of the invention. Any suitable method may be used. In principle, the methods of the invention involve introducing a construct or vector of the invention into a cell, generating a cell of the invention as described above. If the cell is in vivo, for example in a fish, or fish zygote embryo or egg, this leads to a transgenic fish, zygote, embryo or egg of the invention. If appropriate, the fish can be reproduced to produce transgenic progeny fish of the invention. Fish eggs of the invention can be fertilised as appropriate and then grown up to yield fish of the invention, which may then be

reproduced to yield further progeny fish of the invention. Fertilised fish eggs, zygotes and embryos of the invention may be grown up to yield fish of the invention which may then be reproduced to yield further progeny fish of the invention. Progeny fish of the invention may also be reproduced to generate further fish of the invention. Also, fish sperm can be transformed with constructs of the invention and this transgenic sperm can be used to generate transgenic fish of the invention by fertilising fish eggs, optionally transgenic eggs of the invention.

Typically, cells or fish (including eggs, zygotes and embryos) are subjected to suitable selection procedures to separate transformed cells or fish from non- transformed ones.

It is also possible to introduce vectors or constructs of the invention into fish cells in culture, and then incoφorate the cells into the fish, for example into mature or immature fish or zygotes, eggs or embryos, to generate fish of the invention. These are optionally reproduced or grown up as appropriate to generate fish of the invention. Preferably, in the methods of the invention, the construct of the invention, or a part of the construct is chromosomally incoφorated into the fish cell genome, for example using an integrative vector. Where a part of the construct is integrated, it is preferably a part comprising the coding sequence, more preferably a part comprising the coding sequence and flanking sequences (e.g. the promoter) capable of directing its expression in the fish cell. One or more copies of the coding sequence may be integrated into each cell.

The vector or construct of the invention may be introduced into the fish cell at any suitable stage in the fish life cycle. For example, it may be introduced into an unfertilised egg, or a fertilised zygote, or a developing embryo, or into an immature fish or a mature fish. Thus obtained unfertilised eggs may be fertilised to yield zygotes of the invention, which may then be grown up to yield embryos and then fish of the invention. Female fish of the invention may produce transgenic eggs of the invention which can be fertilised, optionally by transgenic male fish of the invention, or sperm derived from such fish. The invention also provides methods for producing recombinant polypeptides of the invention by expressing the polypeptide encoded by the coding sequences of the invention in a cell of the invention, and recovering the polypeptide thus produced

by any means known in the art.

Polypeptides of the invention may be produced and recovered from cells of the invention cultured in vitro. However, it is preferred to produce polypeptides of the invention in vivo in the cells of living fish of the invention, for example cells of mature or immature fish, or their zygotes, eggs or embryos of the invention.

The polypeptide may be obtained by harvesting mature fish, or fish eggs, embryos and zygotes and recovering the polypeptide by any suitable means. Optionally, the polypeptide may be isolated, or substantially isolated, e.g. by one or more isolation steps. Optionally, the polypeptide may be purified, completely or partially, e.g. by one or more purification steps.

In a preferred embodiment, mature female transgenic fish of the invention are produced and allowed to produce eggs which eggs may optionally be fertilised. The eggs are harvested and the polypeptide of the invention is then recovered from the eggs. In methods of this nature, the female fish may be superovulated to enhance the number of eggs produced, and thus the amount of polypeptide recovered. Optionally, in such methods, expression is specifically obtained in the eggs by means of a suitable promoter.

In another preferred embodiment, mature transgenic fish of the invention are generated, and polypeptides are expressed in their tissues. Preferably, expression takes place selectively, in the skeletal muscle, e.g. under the control of a muscle- specific promoter such as the caφ FG2 MyoHC promoter described in Gauvry et al (1996), ibid.

In another embodiment, a DNA or RNA sequence encoding a polypeptide of the invention can be used to achieve transient expression of the polypeptide in a developing fish egg, zygote or embryo. RNA encoding the polypeptide is introduced into a fish egg, optionally a fertilised egg. The egg is, if appropriate, fertilised and grown up to a suitable stage in development, which allows the polypeptide to be expressed.

The polypeptide expressed is recovered by any means known in the art. The polypeptide may be recovered from the eggs at any stage in their development.

The invention will be described with reference to the following Example which are intended to be illustrative only and not limiting. The Example refers to the

Figures. Referring to the Figures in more detail:

Figure 1 is the Invitrogen™ expression vector into which the FVII cDNA is inserted. Figure 2 is the construct from which the FVII cDNA was isolated. Figure 3 is the constnict formed by inserting the FVII cDNA into the expression vector in Figure 1.

Figure 4 is a graph showing catfish embryos expressing recombinant human factor VII.

EXAMPLE

PRODUCTION OF RECOMBINANT HUMAN BLOOD CLOTTING FACTOR-VII IN FISH EMBRYOS

MATERIALS AND METHODS

Plasmids

The DNA encoding human factor VII has been cloned and sequenced (O'Hara et al. (1987) PNAS, Vol 84; 5158-5162 - Genbank Accession No. J02933). The particular human factor VII coding gene cDNA used in these experiments was isolated by Hindlll/BamHI double digestion from the plasmid containing the FVII sequence flanked by a 0.9 kb rat myosin heavy chain gene-derived muscle specific regulatory element (pMyHC/FVII). (Examples of rat myosin heavy chain promoter sequences are published as Genbank Accession Nos. U83321 and U55179). The 2.62 kb FVII cDNA fragment containing the 2.46 kb FVII cDNA and the 0.12 kb SV40 polyA signal sequences was ligated into the pcDNAI expression vector

(Invitrogen™) into the Hindlll/BamHI sites. The resulting plasmid pCMV/FVII (Fig 2) was checked for the appropriate insertion and orientation by restriction mapping. It contains the CMV virus El early promoter and enhancer 5' to the FVII sequences. The plasmid was prepared using conventional molecular techniques (Sambrook et al, (1989), ibid) and used for microinjection into fertilised fish eggs.

Microinjection and embryo culture

Embryos of African catfish were produced by artificial propagation of African catfish broodstock The females were injected with 3 mg/kg body weight caφ pituitary dissolved in PBS solution 8 hours prior to planned ovulation. The eggs were stripped of the females and fertilised by addition of sperm derived from testis surgically removed from males. Single cell embryos (zygotes) were used for microinjection.

Zebrafish embryos were gained from natural spawnings of broodstock kept at 14 hours light 10 hours dark cycle at 28°C. Eggs were late into spawning boxes (Westfield, M (1993) The zebrafish book: A guide for the laboratory use of zebrafish

(Brachydanio rerio), University of Oregon Press, Eugene, OR, USA) and collected from the boxes for microinjection at the early developmental stages (1-2 cell stage).

The pCMV/FVII and the pMyHC/FVII plasmids were both microinjected in circular form into African catfish or zebrafish embryos at the zygote stage in approximately 10 ⁶ to 10 ⁷ copies using a pressure driven picoinjector. The embryos of catfish were placed on Petri dishes where the naturally sticky eggs remained attached. The injection was done by hand using glass microcapillary filled with the appropriate DNA solution containing blue food dye for localisation of injection drop. The zebrafish embryos were placed into an agar gel injection mould designed to hold eggs in row for injection (Westerfield et al, 1993). The injection in case of both species was targeted into the cytoplasmic region or the boundary between the cytoplasmic and yolk regions of the egg. The microinjected embryos were cultured for one day at 28°C in thermostat in Holtfreter's solution until reaching the prim 5 stage (prior to hatching). The microinjected and control (non injected) embryos were collected into microcentrifuge tubes homogenised by plastic homogeniser. The tubes containing up to 1 to 100 homogenised embryos (approx 20 μl) were snap frozen in liquid nitrogen and stored at -70°C until analysis.

RESULTS Immunochemical detection of FVII protein from fish embryo homogenates

ELISA assay was carried out in 96 well plates coated with monoclonal

antibody raised against human FVII (murine monoclonal antibody Clone HVII-1, Sigma), Fish embryo homogenates, dilutions of normal blood plasma containing FVII protein and appropriate negative control samples were loaded into the wells. Biotinylated anti-FVII TAG Antibody conjugated with horse radish perodiase was then added. After addition and incubation with the colour reaction buffer, the samples were read in a conventional ELISA reader. Results of zebrafish and catfish embryos are shown in table I and Fig 4 respectively.

Activity Assay Enzymatic activity of the recombinant factor VII produced in the fish embryos was measured by a chromogenic assay Coaset FVII (Chromogenix, Sweden). The assay is based on a two stage principle. In stage 1 , human factor X is activated (FXa) by thromboplastin in the presence of Ca ²⁺ and FVII in the extrinsic pathway. FVII is completely converted to FVIIa during this process so there is no pre-activated FVII Interference in the assay. In stage 2, the generated FXa hydrolyses the chromogenic substrate S-2765, liberating the chromophoric group pNA. The colour is read photometrically at 405 nm. The generated FXa thus increases the intensity of the colour which is therefore proportional to FVII activity in the sample. The assay was carried out in a microtitre plate which had been pre-coated with a 1% solution of BSA in PBS to prevent non-specific proteins binding during the assay. A standard curve was made from normal blood plasma (pooled from 20 individuals with no thrombotic or bleeding history) and used to prepare a standard curve in the range 200 U/dl to 12.5U/dl by doubling dilutions from 1 :5000. Plasma samples were diluted 1 :1000.

Fish embryo samples were measured undiluted and in doubling dilutions from 1 :40. The data shown in table II was taken from the 1 :1280 dilutions of the samples.

Results were multiplied by 1.28 to match the normal plasma dilution of 1 :1000, and expressed in ng/ml on the basis that 100 U/dl is equivalent to the accepted normal plasma concentration of 2000 ng/ml. Results of activity assay in zebrafish embryos are shown in table II.

Blood clotting assay

Factor VII deficient plasma was used for blood clotting assays (Chanarian, I.

in Laboratory Haematology, Churchhill Livingston, London 1989 pages 286-287) derived from immuno depleted plasma (Diagnostic Reagents Ltd). Thromboplasfin and the appropriate samples were added followed by the addition of CaCl ₂ . The time lapse of the clotting reaction was measured in a Coagulometer KC10. The time lapse results were compared to dilutions of normal plasma samples using a logarithmic scale and were expressed as the percentage activity of normal plasma, where normal plasma means 100 U/dl FVII protein. The results of the blood clotting assay are shown in table III.

Table 1

ELISA test on zebrafish embryos for detection of human FVII protein

Number of FVII concentration embryos (db) (ng/ml) embryos microinjected with pMyHC/F VII: 100 85

100 32 embryos microinjected with pCMV/FVII: 100 186

100 246

1 19 421 control embryos 100 30

FVII dilution series: FVII from FVII concentration serum measured (ng/ml) applied

(ng/ml)

500 508

250 239

125 109

63 65

16 14

0 8

Table II

Activity assay for recombinant FVII produced in fish embryos:

Number of FVII concentration embryos (db) (ng/ml) embryos microinjected with pCMV/FVII: 14 80

100 880

50 540 non injected control embryos 100 0

Table III

Results of bioassay for the blood clotting activity of recombinant FVII produced in fish embryos using FVII deficient blood plasma

Number of % activity * pCMVFVII injected embryos

1. 100 21

2. 100 25

3. 100 28

4. 100 22 5 5.. 5 500 29

non injected control embryos

1. 100

2. 10 control blood plasma 100 bank

* Blood clotting activity is measured in the % of the activity of normal human blood plasma containing normal levels of FVII (100 u/dl).