WO2007026173A1

WO2007026173A1 - Transgenic animal model for identifying modulators of protein kinase

Info

Publication number: WO2007026173A1
Application number: PCT/GB2006/003266
Authority: WO
Inventors: Rune Blomhoff; Harald Carlsen; Jan Øivind MOSKAUG
Original assignee: Birkeland Innovasjon As; Leathley, Anna
Priority date: 2005-09-02
Filing date: 2006-09-04
Publication date: 2007-03-08
Also published as: GB0517977D0

Abstract

The present invention provides a transgenic non-human animal which expresses a bioluminescent signal-generating reporter molecule which has been modified relative to the wild type, or unmodified, reporter molecule to introduce a phosphorylation site such that generation of the bioluminescent signal by the reporter molecule is sensitive to phosphorylation at said phosphorylation site, methods of making such trangsenic animals, and methods of identifying the presence of a modulator of protein kinase using such animals.

Description

87147.627

Transgenic animal model for identifying modulators of protein kinase

The present invention relates to a non-invasive system for identifying protein kinase modulators in vivo. In particular, the present invention provides a system based on a living mammalian animal, such as a mouse, used as a reporter of protein kinase activity. A transgenic animal is provided which carries a gene for a modified reporter protein, modified to be responsive to changes in protein kinase activity levels. Thus, increases or decreases in protein kinase activity in the animal, for example in response to stimulators or inhibitors of protein kinase activity, may turn the reporter protein on or off (specifically phosphorylation of the reporter by protein kinase may activate or inactivate the reporter). The signal generated by the reporter may be detected by an in vivo imaging system. Specifically, the reporter molecule according to the invention generates a bioluminescent signal. Protein phosphorylation is key to the regulation of normal mammalian cell function and its deregulation or abnormal function can be caused by or may cause a wide variety of diseases, including cancer and immune diseases such as rheumatoid arthritis, inflammatory bowel disease, asthma, coronary heart disease and atherosclerosis. In view of the involvement of protein kinases in these disease processes, there is enormous interest in the development of kinase modulators, such as kinase inhibitors to cure diseases or to alleviate disease symptoms and a number of such kinase inhibitors have been identified (Cohen, P. 2002 Nature Reviews Drug Discovery 1: 309-315), several of which have progressed to the clinical trial stage. For example p38MAPK inhibitors are interesting as p38MAPK controls both production of pro-inflammatory agents such TNFα and ILl and their receptor mediated signal transduction, thereby controlling the vicious cycles in inflammation and immune responsive diseases.

Clearly, the identification of further protein kinase modulators or inhibitors would have a significant impact on our ability to treat a wide variety of diseases, and for this reason there is an intense search under way to identify such compounds (English and Cobb, 2002, TiPS 23(1), 40-45). Present methods of identifying and testing these compounds are, however, limited in that they are often performed either in cell free systems or in cell culture systems (i.e. in vitro). This does not always allow for the detection of non-specific effects which many modulators of protein kinases exhibit (Perkel et al. 2002, The Scientist 2: 16(17)) which maybe detrimental to a kinase modulator's chances of success as a drug.

Furthermore, the ability of the potential kinase modulator to reach the appropriate target cells and tissues cannot be tested in these in vitro or cell free systems, as the candidates are applied directly to cells in culture, or even to cell free systems (English etal. 2002, TiPS 23(1): 40-5). When modulators are first identified as being potentially useful using these in vitro or cell free systems, any potential drawbacks such as non-specific effects or lack of bioavailability may only become apparent at a much later stage in the drug development process. For example, a modulator might be identified in vitro, as having an inhibitory effect on a certain kinase, but only after further tests in vitro and in vivo might it become apparent whether or not the modulator will reach the tissue or cell of interest following administration, or whether the modulator has other, specific or non-specific effects on other proteins, or whether the stability of the modulator in vivo is too low for it to be useful as a drug candidate.

It would therefore be advantageous if there were a system which could be used in vivo in a living animal to identify protein kinase modulators in a clinically relevant system. Preferably the system should test the specificity of the candidate protein kinase modulators, together with its ability to reach the target cells and tissues, in vivo.

Despite the importance to our understanding of all aspects of cell biology, progress toward identifying in vivo modulators of specific protein kinases has been slow. Previously used techniques to identify kinase modulators are based on traditional genetic and cell culture systems with phospho-specific antibodies and/or biochemical purifications and radioisotope analysis which often are laborious and unreliable (Manning BD, Cantley LC. Sci STKE. 2002 Dec 10;2002(162):PE49). The in vivo effects of modulators developed by such techniques have most often been disappointing due to the inherent unphysiological characteristics of in vitro systems. The present invention aims to provide a starting point for the discovery and development of selective physiological/pharmacological protein kinase modulators, by providing an in vivo model which allows screening of candidate modulators in a physiologically valid environment.

The inventors have thus designed a new system which allows protein kinase modulators to be identified. As mentioned above, the system is based on an animal model, specifically a reporter animal expressing a modified reporter protein which is sensitive or responsive to protein kinase activity. The animal is thus a transgenic animal carrying a "transgene" (i.e. a foreign or heterologous nucleotide sequence) for a modified, protein kinase-sensitive, reporter protein. The enzyme luciferase traditionally used as a reporter in many systems, may be modified to respond to protein kinase and used according to the present invention. The system has the advantage that it is an in vivo animal-based system and thus modulators that are not suitable for in vivo use are identified early and eliminated. Furthermore, the fact that the system is an in vivo animal system means that the kinase modulators may be assayed for in the context of different animal models of disease. Thus, the kinase- identiflcation (i.e. reporter) system may be provided in an appropriate animal background, for example an animal disease model, either by introducing the modified reporter (i.e. the transgene) directly into the selected animal (i.e. disease model) or by crossing the transgenic reporter animal with available disease models and assaying the kinase modulators in suitably selected progeny. Many different animal disease models are available for performing such assays.

A further advantage of the present system is that the effect of the potential kinase modulators can be assayed directly in vivo in a non-invasive manner, without the need to kill the animal. Thus, the number of animals required for testing is dramatically reduced compared to conventional animal experiments.

The invention makes use of recent developments in in vivo imaging, which make it possible to detect optical signals, particularly bioluminescent signals, in living mammalian animals, e.g. mice.

For example, it has recently been shown that luciferase activity can be detected in vivo and this has been used to monitor NF-κB activation non-invasively (Carlsen et al 2002, The Journal of Immunology, 168: 1441-1446). Detection of luciferase has traditionally been carried out in cell-free systems, by measuring light emission after addition of the luciferin substrate. More recently, as documented in e.g. Carlsen et al, supra, however, bioluminescence in vivo imaging systems based on the expression of luciferase in animals have been developed.

Luciferase is a well-known enzyme which has the ability to produce bioluminescence, which can be detected and measured by various means. It was originally isolated from the firefly, Photinus pyralis, and has the unique property of generating light when a small organic molecule is oxidized at the expense of ATP. The number of photons emitted in this oxidation reaction is proportional to the amount of luciferase when both substrate and ATP are present in excess.

The luciferase substrate is luciferin. In the absence of luciferin, there is no bioluminescence. An advantage of the use of this system for in vivo imaging is therefore that bioluminescence is only produced when the exogenous luciferin substrate is applied or administered.

Any detection of luciferase activity, including in vivo imaging of luminescence, is achieved by administration of luciferin (e.g. by injection). For example, traditionally luciferin has been added to the growth medium of cells expressing luciferase or to a cell lysate made from such cells. As mentioned above, luciferase may be modified and used as a reporter according to the present invention to provide a biomminescent signal for imaging. In the present invention, luciferin must be administered to a transgenic non-human animal. This can e.g. be done by injection into the anaesthetized transgenic non- human animal having the gene coding for the modified reporter luciferase in its genome.

Light emitted from the animal can then be monitored. For example, this can be done with an ultra sensitive camera over a period of time. Pseudo coloured images can then be produced, representing a quantitative measurement of luciferase activity. As mentioned above, it has been shown that this technique monitors the amount of luciferase activity in the animals (Carlsen et al. supra).

The properties of light-generating bioluminescence-producing protein molecules such as luciferase are dependent on their structure, and like many other proteins, the structure and function of these proteins may be modulated by post- translational modifications, such as phosphorylation. For example, in the case of luciferase, the inventors have shown that the introduction of a target site for protein kinase A (PKA) (i.e. RRXS (SEQ ID NO: I)) into luciferase can cause the protein to become responsive to phosphorylation in whole cells and in vivo in living animals and that the changes in the light-emitting properties as a result of the phosphorylation at this site can be detected in vivo in an intact living animal, in particular in a non-invasive manner. As discussed in more detail below, this system is ideally suited to identifying potential protein kinase modulators, and makes use of the fact that the phosphorylation status of the reporter molecule, e.g. luciferase, at the newly introduced phosphorylation site can be readily assayed.

Sala-Newby et a introduced a single point mutation at position 217 of the Photinus pyralis luciferase, which caused the presence of a consensus PKA phosphorylation site. This mutant luciferase, when expressed cytosolically, had a markedly reduced overall luciferase activity in transfected COS cells compared to that of wild type luciferase. A further but modest reduction in luciferase activity (of around 10%) was seen in the mutant V217R protein following the addition of a cAMP analogue to the cells expressing the V217R protein. In order to detect a large effect, in vitro addition of the catalytic subunit of PKA was carried out. This caused an 80-90% decrease in the activity of the mutant luciferase (V217R) compared to its activity in the absence of the catalytic subunit of PKA (Sala-Newby et at. (1992) FEBS 11346, 307(2), 241-244).

In Waud et al (Biochemica et Biophysica Acta (1996) 1292: 89-98), further mutations were made to the luciferase sequence. A sequence equivalent to the PKA kemptide (LRRASLG (SEQ ID NO:2)) or an AMP-PK/cdc2K substrate sequence was introduced at various positions in the protein. All the mutations that were made in the region around 209-227 (which was subsequently identified as being the alpha helix that is involved in ATP binding) led to the production of luciferase mutants in which the enzyme activity and therefore the ability of the luciferase enzyme to generate light was destroyed (levels were less than 0.10% of wild type). These mutants were not investigated further. Several of the mutations that were made to introduce PKA, AMP-PK and cdc-2 phosphorylation sites into the C-terrninus of luciferase however retained some bioactivity. This ranged from 38 to 120%, although only one of these was sensitive to the effect of phosphorylation. Waud et al. demonstrated that one particular mutant having the PKA phosphorylation site at the C-terminus showed reduced activity when 538 U/ml of a PKA catalytic subunit was co-administered to a cell free system. This was demonstrated by expressing and purifying the mutant protein KCT-8 and then measuring the bioluminescence of the KCT- 8 mutant in the presence and absence of the PKA catalytic subunit. The effect of the introduction of a thrombin site was also measured.

This therefore provides a cell-free in vitro system for studying the effects of covalent modification of luciferase at its C-terminus. The system relies on the measurement of luciferase activity in cell-free extracts, and the phosphorylation of the site in luciferase is only observed following the addition of an exogenous catalytic subunit of PKA, and not simply in the presence of a cAMP analogue. The system is of limited application due to the fact that it is carried out in vitro or in a cell-free system.

The inventors have designed a new system which does not share these disadvantages and limitations, and which also has all of the advantages of an in vivo system.

The system relies on the generation of transgenic non-human animals, such as mice, and provides an in vivo system for testing modulators of protein kinase activity.

In a first aspect, the invention therefore provides a transgenic non-human animal which expresses a bioluminescent signal-generating reporter molecule which has been modified relative to the wild type, or unmodified, reporter molecule to introduce a phosphorylation site such that generation of the bioluminescent signal by the reporter molecule is sensitive to phosphorylation at said phosphorylation site.

The transgenic animal thus contains (or carries) a "transgene", or a heterologous (or introduced) nucleotide sequence, which encodes such a modified reporter molecule. As discussed further below, the animal may be capable of expressing an bioluminescent signal-generating reporter molecule as defined above, e.g. under appropriate circumstances. The bioluminescent signal-generating reporter molecule may be any molecule which generates or provides a bioluminescent signal. Bioluminescence is light produced by a chemical reaction within an organism and accordingly a bioluminescent signal-generating reporter molecule is any molecule which mediates such a chemical reaction. The reporter molecule is generally a protein e.g. an enzyme which catalyses such a reaction, preferably a luciferase.

The bioluminescent signal is therefore generated indirectly e.g. by the interaction of the reporter with a further molecule, for example by the action of an enzyme reporter on a substrate. In the case of a reporter protein, the amino acid sequence may be modified to introduce a phosphorylation site.

According to the present invention, the modified reporter is responsive to, or sensitive to, the phosphorylation status of the introduced phosphorylation site. Thus, phosphorylation of the modified reporter at the introduced phosphorylation site may either induce (i.e. cause or produce etc) the optical signal to be generated or may inhibit (e.g. remove or reduce) it. Thus, phosphorylation of the reporter may activate or inactivate the reporter. Preferably, the reporter is inactivated or inhibited by phosphorylation, such that the bioluminescent signal is reduced, or abrogated by phosphorylation. The transgenic non-human animals can therefore be monitored for a bioluminescent signal in the presence or absence of a test compound in order to determine the effect of the test compound on the bioluminescent signal. The bioluminescent signal reflects the phosphorylation state of the reporter molecule in the transgenic non-human animal, and from this information it is possible to determine whether or not the test substance influences phosphorylation of the reporter molecule.

The reporter molecule may be phosphorylated by the action of a protein kinase at the phosphorylation site. Thus, the bioluminescent signal is a reflection, or indication, of protein kinase activity within the animal. If protein kinase is present and/or active in the animal, the reporter will be phosphorylated at the phosphorylation site and may be activated or inactivated by the phosphorylation. If protein kinase activity is reduced or removed in the animal (e.g. through the administration of a kinase inhibitor to the animal) then the reporter will not be phosphorylated at the site (or phosphorylation will be significantly reduced), such that the reporter will conversely be inactivated or activated. It will be understood in this context that "activated" as used herein includes the state of not being inactivated or rendered inactive by phosphorylation; in other words in the absence of phosphorylation (or significant phosphorylation) by protein kinase (i.e. in the absence of protein kinase activity or when protein kinase activity is significantly (i.e. measurably or detectably, or severely) reduced), the reporter remains active. The modified reporter is thus sensitive (or responsive) to protein kinase activity in the animal. The phosphorylation site may thus be viewed as a recognition site, or a target site, for a protein kinase. As discussed further below, different kinases may have different recognition sites and a variety are known and described in the literature, including consensus recognition sequences for particular kinases. An appropriate site may be selected, depending upon the protein kinase(s) it is desired to investigate.

The test compound referred to above may thus be a modulator of protein kinase. This is discussed further below, but such a modulator may be any compound or substance which changes or alters protein kinase activity or amount in any way, and will therefore include an inhibitor or stimulator (or activator) of protein kinase. The system is very flexible as phosphorylation sites are usually only short sequences, the introduction of which may not disrupt the normal reporter molecule function.

By "transgenic" is meant an animal having genetic material artificially inserted into its genome. In other words, the animal comprises heterologous DNA (i.e. a heterologous nucleotide sequence) which has been introduced into the genome. This genetic material may be present as an extrachromosomal element or may be stably integrated into the genomic material in all or a portion of the animal's cells. In general, the transgenic non-human animal will have stable changes to its germline genomic sequence. The transgenic animal may be homozygous or heterozygous for the genetic alteration. Homozygous animals may be bred using standard techniques from heterozygous animals. Techniques for the generation of transgenic animals are well known in the art. A recombinant nucleic acid construct which contains a nucleotide sequence encoding the reporter molecule under the control of an appropriate promoter is first generated. This can be introduced into the pronucleus of fertilised eggs according to one widely used technique (Hogan et al. 1994 Manipulating the Mouse Embryo, 2nd Edition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). The construct to be injected into the fertilised egg must first be in the linear form which is readily carried out, e.g. by digesting the DNA with a suitable restriction endonuclease. The injected, fertilised eggs are then implanted into a foster pseudopregnant mother for the duration of gestation. The offspring are then tested for the presence of the transgene, e.g. using Southern blotting or PCR. These "founder" animals are then bred, firstly to determine whether the transgene is passed on to the offspring, and then to determine whether or not an offspring animal in fact contains the transgene. The founder animals are then bred to homozygosity. Once the transgenic non-human animals have been bred to homozygosity, continued testing for the presence of the transgene is not necessary.

Alternatively, the recombinant nucleic acid constructs containing the nucleotide sequence encoding the reporter molecule under the control of an appropriate promoter can be introduced into a pluripotent cell (embryonic stem cell (ES cell)). If this method is used, a sequence encoding a positive selection marker and regions containing sequences that are homologous to the genomic sequences of the animal (regions of homology) should also be included in the construct.

ES cells are cultured under suitable conditions, and the recombinant targeting constructs are introduced into the ES cells by any method which will permit the introduced molecule to undergo recombination at its regions of homology, for example, micro-injection, calcium phosphate transformation, or electroporation (Toneguzzo, F. et al., Nucleic Acids Res. 16: 5515-5532 (1988); Quillet, A. et al, J. Immunol. 141 : 17-20 (1988); Machy, P. et al, Proc. Natl. Acad, Sci. (U. S. A.) 85: 8027-8031 (1988)). The construct to be inserted into the ES cell must first be in the linear form e.g. by digesting the DNA with a suitable restriction endonuclease. After introduction of the genetic sequences, the ES cells are cultured under conventional conditions and screened for the presence of the construct using known techniques. Cells that survive the selection process are then screened by other methods, such as PCR or Southern blotting, for the presence of integrated sequences.

The selected ES cells containing the construct in the proper location are identified, are inserted into an embryo, preferably a blastocyst, for example by microinjection. The appropriate stage of development of the embryo at which the ES cells are inserted depends on the particular species that is used for generation of the transgenic non-human animal. In mice it is about 3.5 days.

After the ES cell has been introduced into the blastocyst, the blastocyst is typically implanted into the uterus of a pseudopregnant foster mother for gestation. Offspring are then screened using standard techniques known in the art e.g. Southern blots and/or PCR. Mosaic (chimeric) offspring are then bred to each other to generate homozygous animals.

As is the case for transgenic animals generated by pronuclear injection, homozygotes and heterozygotes may be identified e.g. by Southern blotting of equivalent amounts of genomic DNA from animals that are the product of this cross, or with known heterozygotes or wild type animals.

The transgenic non-human animal may be any non-human animal, but is preferably a mammal and more preferably a domestic animal such as a cow, pig, goat, sheep, horse or farmed fish or a laboratory animal e.g. a primate or a rodent such as rat, mouse, hamster, rabbit or guinea pig. Preferably the transgenic animal is a rodent and most preferably a mouse.

The transgenic non-human animal can express a reporter molecule which generates or emits a bioluminescent signal. This signal can be detected using various techniques known in the art. One advantage is that these signals can be detected in vivo, i.e. in non-invasive imaging. This offers the advantage that the transgenic animal can be used in multiple sequential experiments, and it does not have to be sacrificed, e.g. in order to assay for the protein kinase modulator by testing protein kinase activity. Although it is possible, and advantageous, to detect the signal generated from the reporter molecule in a non invasive manner in vivo, it is also possible to sacrifice the test transgenic non-human animal, and to perform the steps necessary for the detection of the reporter molecule in individual cells, organs or tissues which have been dissected out of or removed from the transgenic non- human animal. This can also be done by imaging or else this can be carried out using other standard methods of assay.

The reporter molecule generates a bioluminescent signal. This is generated indirectly, e.g. by the action of the reporter on or with another molecule (e.g. a substrate).

A preferred family of reporter molecules which can be used is the family of luciferases. Light-emitting systems have been known and isolated from many luminescent organisms including bacteria, protozoa, coelenterates, molluscs, fish, millipedes, flies, fungi, worms, crustaceans, and beetles, particularly click beetles of genus Pyrophorus and the fireflies of the genera Photinus, Photuris, and Luciola.

In many of these organisms, enzymes catalyse monooxygenations and utilize the resulting free energy to excite a molecule to a high energy state. Visible light is emitted when the excited molecule spontaneously returns to the ground state. This emitted light is called "bioluminescence" or "luminescence". Luciferases are unique because they produce singlet excited states which are short-lived (<10^~9 s) and preferentially decay emitting light.

The most widely used is the firefly luciferase (Gould SJ and Subramani S, Anal Biochem, 1988 175(1):5-13) . Preferably, therefore luciferase, and especially firefly luciferase is used as the reporter molecule.

'Luciferase' genes are useful reporters according to the present invention because their protein products catalyse the emission of light from a substrate without requiring exogenous illumination (bioluminescence). A variety of such luciferase- encoding genes are known and available. Luciferases are enzymes that emit light in the presence of oxygen and a substrate (luciferin) and which have been used for realtime, low-light imaging of gene expression in cell cultures, individual cells, whole organisms, and transgenic organisms. Such luciferin-luciferase systems include, among others, the bacterial lux genes of terrestrial Photorhabdus luminescens and marine Vibrio harveyi bacteria, as well as eukaryotic luciferase luc and rue genes from firefly species (Photinus) and the sea panzy (Renilla reniformis), respectively (Greer and Szalay, Luminescence, 2002, 17, 43-74). There are many advantages of using luciferase. Firstly, luciferase activity is relatively unstable in vivo and this allows a dynamic assessment of luciferase activity. This is particularly useful if an inducible promoter or a temporally specific promoter is used to express the luciferase. The presence of luciferase activity thus reflects the recent generation of the enzyme. No accumulation of luciferase occurs. There is also the advantage that there is relatively low background bioluminescence in mammals, compared to other widely used reporter molecules. The signal to noise ratio when using luciferase generated bioluminescence is therefore higher than with other reporter molecules.

The limited occurrence of natural bioluminescence in e.g. vertebrates is an advantage of using a bioluminescent signal generating reporter such as a luciferase enzyme as a reporter to monitor molecular events. Because natural bioluminescence is so rare, it is unlikely that light production from other biological processes will obscure the activity of a luciferase introduced into a biological system. Therefore, even in a complex environment, light detection will provide a clear indication of luciferase activity.

Furthermore, the reaction catalyzed by luciferase is one of the most efficient bioluminescent reactions known, having a quantum yield of nearly 0.9. This enzyme is therefore an extremely efficient transducer of chemical energy. An appropriate substrate, luciferin, must be added, but the other substrates required for the bioluminescent reaction, oxygen and ATP, are available within living cells.

The ability to measure luciferase activity in vivo is a further advantage. This was initially demonstrated by Contag et al. (1998, Nature Medicine, 4(2) 245-7). As well as the obvious advantage that fewer transgenic organisms need to be sacrificed, if the activity of the reporter gene can be measured in a non-invasive manner, the use of an in vivo system also allows monitoring of luciferase activity over time. This means that the pharmacokinetic parameters of the potential protein kinase modulator can be determined, and information can be obtained regarding its half-life and rate of metabolism. The potential protein kinase modulator's ability to reach the relevant tissue and cells may also be tested in this system. The rapid turnover of luciferase also means that any unwanted side affects associated with the build up of exogenous protein in a cell or tissue are avoided. The luciferase substrate, luciferin, is administered to the transgenic animal by any appropriate means, e.g. intravenously or intraperitoneally, and the luciferase activity is measured by standard techniques which are known in the art (Contag et al. supra).

The use of luciferase has the further advantage that no bioluminescence is detected in the absence of the addition of exogenous substrate. The exogenous substrate, luciferin, is added to the test system only shortly before carrying out the detection steps. This has the advantage that the luciferin substrate can be applied locally or topically, to regions of interest, or systemically to the whole animal. The term "luciferase" as used herein includes any enzyme or molecule having luciferase activity, defined as the oxidation or oxygenation of a substrate to an oxidised substrate in the present of O₂ with the concomitant generation of light. More particularly, luciferase activity may be defined as the oxidation of a substrate (e.g. luciferin) to an oxidised substrate (e.g oxyluciferin) in the presence of O₂ and ATP with the concomitant generation of light. This substrate may be any substrate but is preferably luciferin. Luciferase catalyses the oxygenation of substrates, generating energy-rich peroxidic intermediates whose spontaneous decomposition generates single electronically excited products which decay emitting a photon of visible light with high efficiency.

The term "luciferase" is used as a generic name to cover any such enzyme having the specified activity because none of the major luciferases share high levels of sequence homology with each other. Luciferases occur in bacteria, fungi, dinoflagellates, radiolarians and about 17 metazoan phyla and 700 genera, mostly marine (Greer and Szalay, Luminescence, 2002, 17, 43-74) including Annelida (segmented worms), Chordata (some elasmobranchiomorphs or sharks, many teleosts or bony fishes), Cnidaria (jellyfishes, anthozoans such as the sea pansy, Renilla), Chaetognaths (one species of arrow-worm), Crustacea (many, including ostracods and euphausiid shrimps or krill), Ctenophora (comb jellies), Echinodermata (sea stars, brittle stars), hemichordate worms, Insecta (fireflies, click beetles), Molmsca (squids, octopods, nudibranchs), Nemertean worms (one species), Pycnogonids (sea spiders), Urochordata (larvaceans, pyrosomes, and one tunicate), millipedes and centipedes. Phylogenetic analyses suggest that luciferin-luciferase systems have had more than 30 independent origins (Greer and Szalay, Luminescence, 2002, 17, 43-74). Both native and synthetic luciferases are included and their derivatives (i.e. luciferases which have been altered from their native or wildtype form, for example by sequence modification, e.g. by recombinant DNA techniques, or by truncation, etc). Active fragments of native or variant luciferases are included, and to be considered "active", the luciferase must simply have the specified activity. Thus, the term includes both prokaryotic and eukaryotic luciferases, as well as variants possessing varied or altered optical properties.

Luciferases have been isolated from various sources. The cDNAs encoding luciferases of various beetle species have been reported. (See de Wet et al, Molec. Cell. Biol 7, 725-737 (1987); Masuda et al, Gene 77, 265-270 (1989); Wood et al, Science 244, 700-702 (1989)). Mutant luciferases of fireflies of the genus Luciola are also known in the art. Kajiyama et al, U.S. Pat. Nos. 5,219,737 and 5,229,285.

The sequences of cDNAs encoding various beetle luciferases, and the amino acid sequences deduced from the cDNA sequences, are known, e.g. LucPplGR (the green-emitting luciferase of the elaterid beetle Pyrophorus plagiophthalamus),

LucPplYG (the yellow-green emitting luciferase of the elaterid beetle Pyrophorus plagiophthalamus ) LucPplYE (the yellow -emitting luciferase of the elaterid beetle Pyrophorus plagiophthalamus), LucPplORK (the orange emitting luciferase of the elaterid beetle Pyrophorus plagiophthalamus) (K. Wood, Ph.D. Dissertation, University of California, San Diego (1989), Wood et al, Science 244,700-702),. Photinus pyralis (Wet et al, MoI. Cell. Biol. 7, 725-737 (1987); K. Wood, Ph.D. Dissertation, University of California, San Diego (1989); Wood et al, Science 244, 700-702 (1989)), Luciola cruciata, Luciola lateralis (Kajiyama etal, U.S. Pat. No. 5,229,285; Masuda et al, U.S. Pat. No. 4,968,613) Luciola mingrelica (Devine et al, Biochim. et Biophys. Acta 1173, 121-132 (1993). Coeloptera sequences are also known e.g. Renilla luciferase (US 5,292,658, US5,418,155). Phengodidae sequences are also known e.g. Phrixothrix luciferase (Viviani et al, Biochemistry 38(26):8271-9, 1999, US Patent application 20050089964).

Sources of different luciferases are thus widely published and it would be a routine matter to isolate a luciferase-encoding sequence from an appropriate source. Further, luciferase-encoding nucleotide sequences are commercially available, for example as plasmids e.g. from Promega, Clontech, Worthington Biochemical Corporation, Mirus, Cambridge Bioscience or Biocompare. Exemplary mention may be made of plasmids of the pGL2, pGL3, phRL or phRG types (all Promega).

Any of these may be chosen for use in the invention. Preferably the luciferase which is modified according to the present invention (i.e. into which the phosphorylation site is introduced) is the Photinus pyralis luciferase and variants thereof (for sequence see: de Wet JR et al. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7870-3). The starting or unmodified luciferase (i.e. the luciferase into which the phosphorylation site is introduced) can be a wild-type or native luciferase such as the luciferases referred to above, i.e. luciferase having an amino acid or encoding DNA sequence that is found in nature, or else they can be a mutant luciferase in which the amino acid sequence of the luciferase, or its encoding DNA sequence, has been altered with respect to the native or wild-type sequence. Several mutant luciferases have been described. These mutant luciferases have been altered relative to the wild type sequence in order to modulate or enhance the luminescent properties of the luciferase, or to remove potentially interfering restriction enzyme sites or genetic regulatory sites from the gene or to improve the codon usage for mammalian cells. Mutant luciferases are described in e.g. US 6,387,675, US 6,552,179, US5,670,356.

In using luciferase expression in eukaryotic cells for biosensing, it is also possible to reduce transport of the luciferase to peroxisomes. Sommer et al., MoL

Biol. Cell 3, 749-759 (1992), have described mutations in the three carboxy-terminal amino acids of P. pyralis luciferase that significantly reduce peroxisome-targeting of the enzyme. The luciferase gene as used in the Examples does not contain the peroxisome signal sequence, but could be modified to introduce it. Thus in a preferred embodiment, the reporter molecule is a mutant luciferase. "Mutant" in this context refers to the fact that the starting luciferase, i.e. before modification to introduce the phosphorylation site, has one or more sequence alterations relative to the wild type luciferase, either at the amino acid sequence level of the luciferase protein, or at the level of the encoding DNA. Thus the luciferase protein may be a mutant, as may the nucleotide sequence (DNA) encoding the luciferase. The modified reporter molecule according to the present invention, may therefore have one or more amino acid sequence changes relative to the wild type luciferase, in addition to the introduction of the phosphorylation site which renders the luciferase sensitive to phosphorylation.

In addition to a mutation made to a luciferase to enhance its function, it is also possible to use a variant of a luciferase in which its amino acid (or encoding DNA) sequence has been altered, without the express intention of improving or modulating the luciferase properties.

The sequence of the reporter molecule is modified or altered relative to that of the wild type reporter molecule. (Thus it is modified to introduce a phosphorylation site and may additionally, if desired, comprise further alterations over the wild-type sequence, as discussed above). In practice this is carried out by modifying the nucleic acid sequence which encodes the reporter molecule in the genetic construct which is used to generate the transgenic non-human animal. This modification can be carried out by standard recombinant DNA technology which is well known to the person skilled in the art, e.g. by introducing point mutations in the DNA sequence encoding the reporter molecule which causes changes to the sequence of the encoded reporter molecule, or by inserting additional nucleic acid sequences encoding one or more additional amino acids. It is also possible to delete one or more codons in the sequence encoding the reporter molecule, for example to remove one or more amino acids from the reporter molecule, if the removal of this amino acid causes a phosphorylation site to be created in the reporter molecule.

These insertions, deletions or mutations can be made at the N or C terminus of the sequence of the reporter molecule, or internally within the sequence. As discussed above in the context of luciferase, these modifications may be the only modifications that are present, which distinguish the modified reporter molecule from the wild type or unmodified sequence of the reporter molecule, or else they may be present in addition to other modifications relative to the wild type sequence of the reporter molecule, which have been introduce to modify properties of the reporter molecule other than its sensitivity to phosphorylation.

The introduction of the phosphorylation site is such that the ability of the reporter molecule to provide its bioluminescent signal is influenced by phosphorylation at the introduced site. Thus, in general, the phosphorylation site that is introduced acts as a switch, such that when the site is phosphorylated, the ability of the reporter molecule to generate bioluminescence is impaired. Alternatively, the phosphorylation site that is introduced may act as a switch, such that when the site is phosphorylated, the ability of the reporter molecule to generate bioluminescence is turned on. Preferably, phosphorylation reduces or decreases or impairs the ability of the reporter molecule to generate bioluminescence. This is similar to phosphorylation in natural organisms which either may activate or inactivate proteins, dependent on the local context. The phosphorylation site therefore has to be introduced at a location within the reporter molecule that is carefully chosen so that in the absence of phosphorylation, the reporter molecule can function, i.e. generates a bioluminescent signal on exposure to the appropriate stimuli.

In view of the strong dependency of these properties on the protein's amino acid sequence, it is preferable that as little sequence modification is made to the wild type or unmodified reporter molecule as possible (which as discussed above in the context of luciferase may be a mutant reporter molecule). Preferably 12, 10 or less, e.g. only 1, 2, 3, 4 or 5 amino acids are changed (i.e. substituted), added or removed relative to the unmodified reporter molecule. In addition, the site should be introduced at a location within the reporter molecule where the presence or absence of phosphorylation is able to influence the ability of the reporter molecule to generate a bioluminescent signal on exposure to the appropriate stimuli. Preferably, only when the site is phosphorylated is this ability impaired. The properties of any modified reporter molecule can be readily tested in vitro prior to generation of the transgenic non-human animal e.g. by using standard assays for the activity of the reporter molecule in the presence or absence of promoters or inhibitors of phosphorylation.

In order to determine where it might be appropriate to introduce the phosphorylation site, the skilled person needs to take into account the known structure-function relationships of the reporter molecule that is being used. The structure and function of reporter molecules such as luciferases have been widely and extensively studied. Molecular structures of these molecules are available to the person skilled in the art and it is possible to use this information to identify suitable locations within a reporter molecule for the introduction of phosphorylation sites which fulfil the above criteria.

Furthermore, it is routine and straightforward to test the effect of the introduction of the phosphorylation site, in the presence and absence of phosphorylation. The appropriate genetic construct may be generated and transfected into cells and the ability of the reporter molecule to generate the bioluminescent signal on exposure to the appropriate stimuli in the presence or absence of phosphorylation can readily be determined, e.g. using standard techniques as described in Example 1.

Reference to the introduction of a phosphorylation site indicates that a phosphorylation site is present in the modified reporter molecule which is not present in the unmodified reporter molecule. It does not mean that there are no amino acid sequences present in the unmodified reporter molecule that could be modified by phosphorylation. Most phosphorylation sites are quite short (e.g. 3 amino acids in length) and as such it is likely that the unmodified reporter molecule contains stretches of amino acids which could under certain circumstances become phosphorylated. In the reporter molecule this may be prevented under normal conditions e.g. by steric factors.

Thus, the modified reporter molecule comprises or contains a phosphorylation site that is not present in the unmodified reporter molecule. The site may be introduced by changing (e.g. adding, substituting or deleting) one or more amino acids at a suitable location within the amino acid sequence of the reporter molecule (one example of this is changing the valine at position 217 to an arginine in firefly luciferase, as in Example 1, so that a PKA site is introduced (RRFS (SEQ ID NO:3), whereas no such phosphorylation site is present in the unmodified reporter molecule (VRFS (SEQ ID NO:4)). Thus in a preferred embodiment, the sequence of the reporter molecule is changed by the substitution of one or more amino acids so as to introduce a phosphorylation site into the reporter molecule and result in a luciferase comprising a phosphorylation site that is not present in the unmodified reporter molecule.

Alternatively, a sequence corresponding to a phosphorylation site may be introduced into the reporter molecule at an appropriate location by changing the nucleotide sequence of the DNA encoding the reporter by inserting one or more amino acids in the construct which is used to generate the transgenic non-human animal.

The introduced phosphorylation site is selected depending on the protein kinase which is to be studied or investigated e.g. for which modulators are being sought. As protein kinase phosphorylation sites are known and well-characterised, a suitable site for the phosphorylation by the protein kinase whose activity is to be investigated or modulated may be selected and introduced into the reporter molecule. The effect of a potential modulator on the protein kinase may thus be detected indirectly i.e. by virtue of its influence on the reporter molecule e.g. its luciferase activity.

Phosphorylation may occur at serine, threonine or tyrosine residues, depending on the phosphorylation site and the particular kinase. There are over 300 kinase proteins, which share sequence similarity and protein structural motifs important for their function. The knowledge of the phosphorylation sequence for those kinases enables the skilled person to modify the sequence of the kinase so as to cause the insertion of an appropriate site into the reporter molecule, depending on the particular protein kinase to be studied (e.g. for which modulators are being sought).

The phosphorylation site that is introduced can be a site for a tyrosine kinase, and protein tyrosine kinases can be part of a receptor or may be soluble. In one embodiment therefore, the protein kinase may therefore be a protein tyrosine kinase, in which case the introduced phosphorylation site is preferably a sequence comprising or consisting of (K/R) X X (D/E) X X X Y (SEQ ID NO:5) or KTR X X X (D/E) X X Y (SEQ ID NO:6), where X is any amino acid. Examples of receptor tyrosine kinases are growth factor receptors such as the

EGFR, c-Kit receptor, VEGF receptor, c-Met receptor, insulin growth factor receptor, and Eph receptor. Examples of cytosolic tyrosine kinases are src and src related kinases and janus (JAK) kinases.

Alternatively, the phosphorylation site that is introduced maybe a site which is phosphorylated by a serine or threonine kinase. Examples of serine/threonine protein kinases are phosphorylase kinase (GPK), pyruvate dehydrogenase kinase, cAMP dependent protein kinase (PKA), cGMP dependent protein kinase (PKG), protein kinase C (PKC), Ca²⁺/calmodulkι-deρendent protein kinases, G protein coupled receptor kinase (GRKs), Mitogen-activated Protein, various oncogenes (including mil, raf and mos), receptor serine/ threonine protein kinases (TFGβ super family).

One preferred example is the phosphorylation site for PKA. The PKA protein phosphorylates peptides that contain the sequence R/L R/L X S/T (SEQ ID NO: 7), and the reporter molecule may be modified such that it contains this sequence, resulting in a luciferase comprising this sequence. X is any amino acid. Preferably the sequence can be defined as R/L R/L X₁ S/T X₂ (SEQ ID NO:

8) wherein X₁ is any amino acid, preferably a small amino acid (e.g. glycine, alanine) and X₂ is a hydrophobic amino acid (e.g. valine, isoleucine, leucine, methionine, phenylalanine, tryptophan and cysteine). Even more preferably the sequence is RRXj S. Thus in a preferred embodiment, the reporter molecule is modified to contain or comprise one of the above-described sequences.

In another preferred embodiment, the phosphorylation sequence for PKC is introduced into the reporter molecule. The phosphorylation sequence for PKC can be defined as T/S X R/K (SEQ ID NO:9), but different studies suggest variations to this PKC consensus target sequence. One preferred phosphorylation sequence for PKC is TXR. It would be a routine matter, if desired, to optimise the position of and/or the sequence of the PKC target that retains luciferase activity.

Further protein kinase phosphorylation sites are identified below in Table 1 , together with examples of phosphorylation sites that fit the recognition motif. Any of these recognition motifs or phosphorylation sites may be introduced into the reporter molecule.

Where a sequence is identified as a recognition motif or phosphorylation site, it should be understood that modifications to the luciferase which are to introduce a recognition motif or phosphorylation site include the introduction of a sequence that comprises such a recognition motif or phosphorylation site. The modified luciferase as a whole thus comprises one or more recognition motifs and/or phosphorylation sites that are not present in the unmodified molecule.

Table 1 Protein Kinase Recognition Motif Phosphorylation Site

Casein Kinase I S(P)-X-X-S/T R TLS(P)VSSLPGL (SEQ ID NO: 11) (SEQ ID NO: 10) DIGS(P)ES(P)TEDQ (SEQ IDNO: 12)

Casein Kinase II S/T-X-X-E ADSESEDEED (SEQ ID NO: 14) (SEQ ID NO: 13) LESEEEGVPST (SEQ ID NO: 15) EDNSEDEISNL (SEQ ID NO: 16)

Glycogen synthase 3 S-X-X-X-S(P) SVPPSPSLS(P) (SEQ ID NO: 18) (SEQ ID NO: 17) SVPPS(P)PSLS(P) (SEQ ID NO: 19)

Cdc protein kinase; S/T-P-X-R/K PAKTPVK (SEQ ID N0:21) CDKZ-cyclin A (SEQ ID NO:20) HSTPPKKKKRK (SEQ ID NO:22)

Calmodulin-dependent R-X-X-S/T(SEQ ID NO:23) NYLRRRLSDSN(SEQ ID NO:24) Protein Kinase II R-X-X-S/T-V(SEQ ID NO:25) KMARVFSVLR(SEQ ID NO:26)

Mitogen activated P-X-S/T-P(SEQ ID NO:27) PLSP(SEQ ID NO:2δ) Protein kinase X-X-SZT-P(SEQ ID NO:29) PSSP(SEQ ID NO:30)

(Extracellular signal related kinase)(MAPK, ERK)

In the table above, S(P) indicates a phosphorylated serine residue. Preferably, the phosphorylation site is a MAP kinase phosphorylation site.

Examples of such phosphorylation sites are set out in Table 2. Table 2 - Members of the MAPK family

MAPK subtype Other names P-site motif

ERKl PKl, p44 MAPK TEY

ERK2 MAPK2, p42 MAPK TEY

ERK3 - SEG

ERK5 BMKl TEY

ERK7 - TEY

JNKl SAPKlγ TPY

JNK2 SAPKl α TPY

JNK3 SAPKl β TPY p38α SAPK2a, CSBP TGY

_P39β SAPK2b TGY p38γ SAPK3, ERK6 TGY p38δ SAPK4

MAK male germ cell TDY associated kinase

MRK MAK related kinase TDY

MOK - TEY

KKIALRE - TDY

KKIAMRE — TDY

Thus in a preferred embodiment, the introduced phosphorylation site is TX₆Y (SEQ ID NO:31), wherein X₆ is E, P, D or G or SEG (SEQ ID NO:32). Preferably the protein kinase is ERKl , 2, Ib, 5 or 7 or MOK and the introduced phosphorylation site is TEY. Alternatively the protein kinase is JNKl, 2 or 3 and the introduced phosphorylation site is TPY.

In a further alternative the protein kinase is p38α, β, β2, γ or δ and the introduced phosphorylation site is TGY or the protein kinase is MAK, MRK, KKIALRE or KKIAMRE and the introduced phosphorylation site is TDY In a further alternative, the protein kinase may be a member of the MEK family (see Table 3).

Table 3 - Members of the MEK family

MEK subtype Other names P-site motif

MEKl MKKI₅ MAPKKI SMANS

MEK2 MKK2, MAPKK2 SMANS

MEK3 MKK3, SKK2 SVAKT

MEK4 MKK4, JNKKl, SEKl, SKKl SIAKT

MEK5 MKK5 SIAKT

MEK6 MKK6, SKK3 SVAKT

MEK7 MKK7, JNKK2 SKAKT

Thus the introduced phosphorylation site may be S X₃A X₄ X₅ (SEQ ID

NO:33) where X₃ is M, V, I or K Xds N or K X₅ is S or T. Preferably the introduced phosphorylation site is SMANS and the protein kinase is MEKl or MEK2. Alternatively the introduced phosphorylation site is SVAKT and the protein kinase is MEK3 or 6. In a further alternative, the introduced phosphorylation site is SIAKT and the protein kinase is MEK4 or 5. The introduced phosphorylation site may also be SKAKT and the protein kinase is MEK7.

As mentioned above, the literature is replete with references to and descriptions of protein kinases and their recognition sites. It would be a matter of routine to design an appropriate recognition site for a desired protein kinase or group of protein kinases, based on this information. Consensus sequences for recognition sites for particular kinases have also been published, and could readily be assembled using available information. As mentioned above, the phosphorylation site may be introduced by making point mutations or by adding in, i.e. inserting into the reporter sequence additional residues, in which case the inserted sequence may comprise the phosphorylation site, may consist of the phosphorylation site or may consist of a portion of the phosphorylation site which is introduced at an appropriate location within the reporter molecule so as to generate a new phosphorylation site. The mutations or additions can be carried out by standard recombinant DNA technology.

The skilled person must select the location of the introduced phosphorylation site such that when phosphorylation occurs, the bioluminescent signal generating properties of the reporter molecule are affected, i.e. the reporter molecule is sensitive to phosphorylation at the introduced phosphorylation site. Phosphorylation at the introduced phosphorylation site may increase or decrease the degree or intensity of the bioluminescent signal. Preferably the degree or intensity of the optical signal is reduced by phosphorylation at the introduced phosphorylation site. By this it is meant that the reporter molecule behaves in a measurably different way when it is phosphorylated than when it is unphosphorylated. In other words, it is possible to determine from the behaviour of the reporter molecule whether or not it is phosphorylated.

The determination of the phosphorylation status can be achieved by comparing the behaviour of the reporter molecule in the presence or absence of a known stimulator or inhibitor of the relevant protein kinase (i.e. a protein kinase to which there is a phosphorylation site introduced into the reporter molecule). Various stimulators and inhibitors of protein kinase function are known. These may be specific to the protein kinase in question or else they may be general. For example, forskolin is a known stimulator of PKA, and cAMP analogues such as

8CPT-cAMP may also be used to stimulate PKA phosphorylation. An example of a known PKA inhibitor is the compound H89 (Calbiochem (Bad Soden, Germany)) The bioluminescent signal generating properties of the reporter protein are measured by techniques which are well known in the art (Contag et al. supra, Ntziachristos V, Bremer C, Weissleder R Eur Radiol. 2003 Jan;13(l):195-208). Although it is an advantage of the present invention that as described in Contag et al., (supra) the properties of the reporter molecule can be detected in vivo in a non- invasive manner, if necessary, the appropriate tissues or cells can be removed from the transgenic animals and either the cells or tissue can be imaged or else lysates can be made and the biolummsecent signal generating properties of the reporter protein can be measured directly in a cell free system (e.g. by luminometry for luciferase, as described in Example 1).

The bioluminescent signal generating properties of the reporter protein may be increased by phosphorylation at the introduced phosphorylation site, e.g. by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150% or 200% relative to the optical signal generating (e.g. bioluminescent or light emitting) properties of the reporter protein in the unphosphorylated state. It is preferred, however, that the optical signal generating (e.g. bioluminescent or light emitting) properties of the reporter molecule are reduced, e.g. by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150% or 200% relative to the bioluminescent signal generating properties of the reporter protein in the unphosphorylated state. The bioluminescent signal generating properties of the reporter molecule, once phosphorylated may therefore be reduced to less than 80%, 60%, 40%, 20%, 10%, 5%, 2%, 1%, 0.5%, 0.1% or 0.01% of the bioluminescent signal generating properties of the reporter molecule in the absence of phosphorylation. Not all parts of the reporter molecule will be available for phosphorylation by protein kinases, due to the fact that proteins fold up such that certain regions will be buried inside the protein. Other regions of the reporter molecule will however be accessible for phosphorylation by protein kinases.

Crystal structures, NMR structures and other structural data are available for many such reporter molecules and the availability of such data will allow the skilled man to identify regions of the reporter molecule into which a phosphorylation site might usefully be introduced.

It is also important that the phosphorylation site is introduced at a location within the reporter molecule that renders the reporter molecule sensitive to phosphorylation. It is clear that this will not be the case for all locations within a reporter molecule. Again, the understanding of the structure function relationship of such reporter molecules will assist the skilled person in determining the optimal location for the introduction of a phosphorylation site.

Preferred locations for the introduction of the phosphorylation site are domains of the reporter molecule which are involved in bioluminescent signal generation.

The location of the introduced phosphorylation site should also be chosen so the reporter molecule retains its ability to function as such.

It is not necessary that the introduction of the phosphorylation site has no effect on the ability of the reporter molecule to generate bioluminescent signals. What is required is that the reporter molecule has the capacity to generate a bioluminescent signal after the introduction of the phosphorylation site.

Preferably therefore the introduction of the phosphorylation site into the reporter molecule is at a location which renders the reporter molecule sensitive to phosphorylation, but which does not in itself impair the ability of the reporter molecule to function as such. It would not be possible to determine the effect of phosphorylation on the reporter molecule if the reporter molecule's sequence were to be altered such that it could not under any circumstances generate a bioluminescent signal.

It is possible that the introduction of the phosphorylation site into the reporter molecule will either increase or decrease the ability of the reporter molecule to generate a bioluminescent signal , independently of any effect that phosphorylation has on this ability. The introduction of the phosphorylation site into the reporter molecule can either increase or decrease the ability of the reporter molecule to generate a bioluminescent signal. This can be controlled for in any experiment which requires an assessment of the bioluminescent signal generating properties of the reporter molecule, e.g. by comparing the bioluminescent signal generating properties of the reporter molecule in the presence or absence of known phosphorylation inhibitors and activators. Thus, the introduction of the phosphorylation site into the reporter preferably does not increase or decrease the bioluminescent signal generating properties of the reporter molecule relative to the wild-type or unmodified sequence in the absence of phosphorylation, by more than 50%, preferably 40%, 30%, 20%, 10% or 5%. The likelihood of a particular phosphorylation site influencing the bioluminescent signal generating properties of the reporter molecule in the absence of phosphorylation can be predicted using the known structural data available for the various reporter molecules and assessed by measuring the bioluminescent signal generating properties of the reporter molecule in a sample in vitro system e.g. as described in Example 1.

When the reporter molecule is luciferase, it is preferred that the introduced phosphorylation or kinase site is introduced in a region that is accessible for an enzyme, a hinge region between two functional sites (e.g. the sequence corresponding to the flexible loop of P. pyralis luciferase (amino acids 436-440)), or in a functional domain. Examples of functional domains are the luciferin binding site (which is made up of several non-contiguous residues between residues 218 and 348 of the Photinus pyralis luciferase sequence or the equivalent residues in other luciferase molecules), or in the ATP binding domain (which is made up of several non-contiguous residues between 316 and 362 of the Photinus pyralis luciferase sequence, or the equivalent residues in other luciferase molecules), preferably in an α helix comprising part of the ATP binding domain.

In a preferred embodiment the phosphorylation site is inserted at residues 217-220 or 217-224 of the P pyralis firefly luciferase sequence, or the equivalent residues in another luciferase molecule, e.g. residues 216-221, 215-222, 214-223 , 213-224, 212-225 or 210-230.

The P. pyralis firefly luciferase shows a topology such that a main N- terminal (amino acids 1-436) domain is connected by a flexible loop (amino acids 436-440) to a smaller C-terminal domain (amino acids 440-550). The phosphorylation site may be inserted into any one of these domains or the equivalent domains in other luciferases.

The main N-terminal domain consists of a compact domain containing a distorted antiparallel beta-barrel and two βsheets flanked by αhelices forming an ababa five-layered structure. The phosphorylation site may be inserted into any one of these helices or sheets or the equivalent locations in other luciferases.

The C-terminal consists of an a+ b structure, and the phosphorylation site can also be inserted into this region, e.g. between residues 537-550 of the P. pyralis firefly luciferase, preferably at residues 544-550, 540-546, 539-545, 538-544, 537- 543, 543-547, 542-546, 541-545, 540-544 or 539-546 or the equivalent residues in other luciferases,

The surfaces of the N- and C-terminal domain facing each other form a cleft where many conserved residues are found and which is considered to be the active site. During the bioluminescence reaction, firefly luciferases undergo considerable conformational change and the N- and C-terminal domains are likely to come close enough to sandwich the substrates (Viviani CMLS, Cell. MoI. Life Sci, 59(11): 1833-50, 2002). The reporter molecule is preferably luciferase having the sequence

RRFSLRRD (SEQ ID NO:34) at residues 217-224 of the Photinus pyralis luciferase sequence, or the equivalent residues in other luciferase molecules.

In a most preferred embodiment, the reporter molecule is luciferase having the sequence RRXS (SEQ ID NO:1), preferably RRFS (SEQ ID NO:3) at residues 217-220 of the Photinus pyralis luciferase sequence, or the equivalent residues in other luciferase molecules.

The firefly luciferase used in the Examples herein is comprised of two ligated fragments, one from pTAL-luc and one from pGL3 -control, both of which are publicly available plasmids whose sequences are known. The luciferase sequence in pTAL-luc differs from accession number Ml 5077 (submitted as Photinus pyralis luciferase gene by S. Subramani) in two amino acids.

The influence of the introduced phosphorylation site on the bioluminescent signal generating properties of the reporter molecule can be determined using in vitro assays prior to generation of the transgenic animal. It is important to note that the introduction of the phosphorylation site may well influence the bioluminescent signal generating properties of the reporter molecule, independently of the influence on the bioluminescent signal generating properties of the reporter molecule by the presence or absence of phosphorylation. For example, the bioluminescent signal generating properties may be reduced or increased relative to the wild type or unmodified reporter molecule sequence. This does not affect the validity of the experimental approach. What is important is that the bioluminescent signal generating properties are sensitive to phosphorylation. The comparison of the bioluminescent signal generating properties of the reporter molecules must therefore be made in the presence and absence of phosphorylation; the wild type or unmodified sequence is not the appropriate control.

The transgenic non-human animal can be used to identify modulators of protein kinase function. As such, there is also provided the use of the transgenic non-human animal as described and defined herein in identifying modulators of protein kinase function. This is described in more detail below.

In a further embodiment of the invention, there is provided a method of making a transgenic non-human animal of the invention, (i.e. a transgenic non- human animal which expresses an bioluminescent signal generating reporter molecule, which has been modified relative to the wild type or unmodified reporter molecule to introduce a phosphorylation site such that the bioluminescent signal generating properties of the reporter molecule are sensitive to phosphorylation at the said phosphorylation site), said method comprising the step of introducing into said animal (more particularly, into the genome of said animal) a nucleotide sequence encoding an bioluminescent signal generating reporter molecule, which has been modified relative to the wild type, or unmodified reporter molecule to introduce a phosphorylation site such that the bioluminescent signal generating properties of the reporter molecule are sensitive to phosphorylation at the said phosphorylation site. Such a method may optionally further include the step of crossing such an animal with another animal or breeding progeny from such an animal.

The method may thus comprise the steps of introducing a recombinant genetic construct encoding an bioluminescent signal generating reporter molecule, which has been modified relative to the wild type, or unmodified reporter molecule to introduce a phosphorylation site such that the bioluminescent signal generating properties of the reporter molecule are sensitive to phosphorylation at the said phosphorylation site under the control of a promoter into the pronucleus of a fertilised egg, and implanting said egg into a psuedopregnant foster mother. Alternatively the method may comprise the steps of introducing a recombinant genetic construct encoding a bioluminescent signal generating reporter molecule, which has been modified relative to the wild type, or unmodified reporter molecule to introduce a phosphorylation site such that the bioluminescent signal generating properties of the reporter molecule are sensitive to phosphorylation at the said phosphorylation site under the control of a promoter into an ES cell, introducing said ES cell into a blastocyst and implanting said blastocyst into a pseudopregnant foster mother. In this method, it is possible to screen the ES cells for integration of the construct, in which case a positive selection marker should be included in the genetic construct.

In such methods, it would be a matter of routine to select a suitable or desired promoter or an appropriate pseudopregnant foster mother or appropriate ES cell. The recombinant genetic constructs that are used to generate the transgenic non-human animals will generally include the following components: a polynucleotide encoding the reporter molecule, and a suitable promoter operably linked to the reporter molecule. If the transgenic non-human animal is to be made using homologous recombination in ES cells, it is necessary also to include a sequence encoding a positive selection marker, and homologous insertion sequences (Capecchi MR, Trends in Genetics, 1989 5(3):70-6). Insulator sequences, as described in US 5,610,053 can also be included.

Positive selection markers include any gene which encodes a product that can be assayed. Commonly used examples include the hprt gene (Littlefield, J. W., Science 145: 709-710 (1964)) and the TK gene of herpes simplex virus (Giphart- Gassler, M. et al,, Mutat. Res. 214: 223-232 (1989)) or other genes which confer resistance to amino acid or nucleoside analogues, or antibiotics. Addition of the appropriate substrate of the positive selection marker can be used to determine if the product of the positive selection marker is expressed. The transgenic non-human animal may express the reporter molecule ubiquitously, i.e. in every cell of the transgenic non-human animal. Alternatively, the expression of the reporter molecule may be restricted to a particular cell or tissue type, in which case the expression pattern is cell or tissue specific and is spatially regulated within the transgenic non-human animal. In addition to or alternatively to the above, the expression of the reporter molecule may be regulated temporally, so that expression only occurs at a particular time during the development of the transgenic non human animal, or else at one or more particular times during the life of the transgenic non human animal.

Expression of the reporter molecule can also be inducible, in other words the expression of the reporter molecule can be switched on or off, depending on the local conditions in the cell. These conditions can be manipulated artificially, e.g. by addition of inducer molecules to the transgenic non-human animal.

It can be seen that the expression of the reporter molecule can be varied, and this may depend, for example, on the particular type of modulator of protein kinases that is being sought. The expression pattern of the reporter molecule depends on the choice of promoter system for generating the transgenic animal. A large number of different promoters are known which can be used to drive expression of the reporter molecule and it is simply the case of using an appropriate promoter to make the genetic construct which is then used to generate the transgenic non-human animal. The choice of promoter will depend on the particular application of the transgenic non-human animal. If it were desired to generate a single transgenic non- human animal model, which can be used to identify modulator of protein kinase in general, then it would be advantageous to use a promoter which is ubiquitously expressed in the transgenic non-human animal. The generated transgenic non- human animal would then be useful in a broad range of applications. Examples of such promoters are the CMV promoter, ROS A26 promoter and ubiquitin C promoter, (Takada et al, Nature Biotechnology, 15(5):458-61, 1997, Kisseberth, et al Dev Biol, 214(l):128-38, 1999, Schorpp et α/, NAR, 24(9):1787-8, 1996)

Under other circumstances it may be advantageous to only express a reporter molecule in a more restricted manner. For example, it could be disadvantageous to express high levels of an exogenous reporter molecule in every cell of the transgenic non-human animal, as it is possible that this could interfere with the normal functioning of the cells or tissues of that transgenic non-human animal. So, if a modulator of protein kinase is being sought which is effective in a particular tissue, it may be appropriate to generate a transgenic non human animal according to the invention which expresses the reporter molecule only or mainly in that cell or tissue, e.g. in the liver. Other examples of tissues in which tissue specific expression maybe advantageous are the pancreas, mammary gland, epithelium, small intestine, skeletal muscle, smooth muscle, striated muscle, heart, prostate, adipose tissue, neural crest, brain, kidney and lung. Tissue specific promoters have been identified for each of these tissues.

Table 4

Inducible promoters could also be used. These promoters have the advantage that they can be activated or induced to express the reporter molecule. The reporter molecule is thus only expressed when it is necessary for the purposes of the experiment. Examples of inducible promoters are well known in the art and include Cre-, estrogen-, retinoic acid responsive element containing promoters and tetracycline responsive promoters (for a recent review see Albanese C, Hulit J, Sakamaki T, Pestell RG.Semin Cell Dev Biol. 2002 Apr; 13 (2): 129-41)

Examples of promoters which are specific for a certain time during the development of a transgenic non-human animal are the MMTV-LTR, which drives expression during mammary gland development during pregnancy and lactation in the mouse.

The invention also relates to the use of the transgenic non-human animal of the invention in an assay for identifying protein kinase modulators. Thus in a further embodiment, the invention relates to a method for identifying a modulator of protein kinase function, preferably an inhibitor of protein kinase function (e.g. identifying the presence of a modulator of kinase function in a test sample), said method comprising the steps of: a) administering said modulator of protein kinase function (or a test sample containing said modulator of protein kinase function) to a transgenic non-human animal as defined herein, and b) assessing the modulation of protein kinase function by comparing the behaviour of the reporter molecule in the presence and absence of said modulator of protein kinase function (or a test sample containing said modulator of protein kinase function). By use of the term "comparing the behaviour" it will be understood that this includes comparing the bioluminescent signals generated by the reporter in the presence and absence of the said putative modulator (i.e. test compound).

This method can therefore be seen to provide an assay for modulators of protein kinase function e.g. novel modulators of protein kinase function. This includes both novel compounds or entities and the identification or screening of known or existing compounds for the property of modulation of protein kinase function.

By "modulator of protein kinase function" is meant any compound or entity that is able to affect the kinase activity of a protein kinase, i.e., the ability of a protein kinase to phosphorylate its target substrate. This includes all entities or substances that are capable of directly or indirectly affecting the function of the kinase. This may be achieved by affecting the transcription, translation, post- translational modification, activity or regulation of the protein kinase, with synthesis. Preferably the enzyme activity i.e. the ability to phosphorylate the substrate is affected, e.g. by binding to the active site of the kinase (an antagonist), or elsewhere on the kinase such that it cannot perform its normal function. As described further below the modulator may be a stimulator (or activator) of protein kinase, or it may be an inhibitor. The modulator of protein kinase function may be any chemical entity. For example, it could be another protein, or a peptide, a small molecule, e.g. a small organic molecule, antibody, or antibody fragment or derivative, ribozyme, antisense RNA or DNA, siRNA, PNA or an analogue of the substrate. The modulator of protein kinase function may be naturally derived or it may be synthetic. The modulator of protein kinase function may increase or decrease the activity of the relevant protein kinase. Preferably the modulator of protein kinase function decreases the activity of the relevant protein kinase and is termed an inhibitor. The inhibition may result in less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1%, 0.5% or 0.1% of normal activity. If the modulator of protein kinase function increases the activity of the relevant protein kinase, the increase is preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150% or 200% of normal activity. The activity of the reporter molecule may be assayed using any method which is known in the art. Preferably, the activity of the reporter molecule is assayed non-invasively in vivo, particularly using an in vivo imaging system based upon detection of the bioluminescent signal from the reporter, although it is possible also to sacrifice the transgenic non-human animal and remove from said animal the cells, tissues or organs of interest. The activity of the reporter molecule can be imaged in these cells, tissues or organs, or else can be assayed for directly, using techniques known in the art.

Thus, conveniently, an imaging device may be used, particularly an ultra- sensitive imaging device.

The activity of the reporter molecule can be expressed quantitatively (e.g. for luciferase see Contag et al supra. Sophisticated digital camera systems are available which allow direct quantitation of the emitted bioluminescent signal. Pseudo coloured images can be produced which represent quantitative measurement of the activity of a light emitting reporter molecule.

The monitoring of expression of luciferase reporter expression cassettes using non-invasive whole animal imaging has been described (US 5,650,135, Contag, P., et al, supra). Such imaging typically uses at least one photo detector device element, for example, a charge-coupled device (CCD) camera. Methods of monitoring the expression of the reporter molecules in a non invasive in vivo system are described in detail in US5650135 and Zhang et al., J. Immunol Jun 15;170(12):6307-19 2003).

Imaging of the light-emitting entities involves the use of a photodetector capable of detecting extremely low levels of light-typically single photon events-and integrating photon emission until an image can be constructed. Examples of such sensitive photodetectors include devices that intensify the single photon events before the events are detected by a camera, and cameras (cooled, for example, with liquid nitrogen) that are capable of detecting single photons over the background noise inherent in a detection system. Once a photon emission image is generated, it is typically superimposed on a

"normal" reflected light image of the subject to provide a frame of reference for the source of the emitted photons (i.e. localize the light-emitting conjugates with respect to the subject). Such a "composite" image is then analysed to determine the location and/or amount of a target in the subject. The light emission is typically expressed as photons per second per cm per steradian (P/s/cm /sr), which is the intensity of the light escaping the animal. Recently developed technology allows a more detailed interpretation of the image using algorithms to calculate the depth of the light source based on images taken from different angles of the animal, knowledge about properties of light absorption and penetrance in an animal tissue.

These methods will allow the identification of modulators of protein kinases which affect phosphorylation at a particular phosphorylation site, i.e. the introduced phosphorylation site. These modulators may be general modulators in that they affect a broad spectrum of kinases, i.e. they do not only affect kinases which act at the introduced phosphorylation site, or the modulators may be specific to the introduced phosphorylation site.

The modulator of protein kinase function is preferably a specific inhibitor or activator of protein kinase function. By specific inhibitor or activator it is meant that the inhibitor or activator acts only, or preferentially or selectively on the protein kinase or class of protein kinases that phosphorylate a particular phosphorylation site.

As the phosphorylation site is inserted in isolation into the reporter molecule, it is only possible by this method, to identify an activator or inhibitor of a protein kinase that acts at a particular phosphorylation site. For example, ERKl, ERK2 and ERK 7 all phosphorylate the threonine of the TEY motif. Introduction of this motif into a reporter molecule according to the invention and as discussed elsewhere herein will allow the method of the invention to be carried out such that modulators of any kinase which phosphorylates at this site can be identified, by virtue of the fact that the activity of the reporter molecule is sensitive to phosphorylation at this motif, and the presence of a modulator will change the properties of the reporter molecule, when compared to a reporter molecule which has not been exposed to such a modulator. The specificity of the modulator of kinase activity is thus determined in the method of the invention with respect to the phosphorylation motif, and not with respect to the particular protein kinase, unless it is the case that the phosphorylation motif is exclusive to this particular protein kinase.

Many protein kinases act in cascades, for example MEK kinases phosphorylate members of the MEK family (e.g. MEK, MKK, SEK, SKK and JNKK (Pearson et al supra), which in turn phosphorylate ERK/MAPKs. In nature this serves to generate signal amplification and this occurs if each successive protein in the cascade is more abundant than its regulator.

The knowledge of the existence of such a cascade can be utilised in the method of the invention. For example, Raf (and other proteins) can phosphorylate MEKl and 2, which in turn phosphorylate ERKl and 2. If it were of interest to identify a modulator of protein kinase activity which acts on this pathway, then it would be possible to choose between the introduction of a phosphorylation site for raf, a phosphorylation site for MEKl or 2 [SMANS], or a phosphorylation site for MAPK [TEY] as inhibition or activation at any level of the pathway will be reflected in the activity of the reporter molecule.

The choice of site to introduce will thus depend on the nature of the activator or inhibitor that is required. The activity of the modulator is not measured directly, e.g. by measuring the activity of the kinase in a kinase assay, as has been done previously, but instead it is measured functionally, in that the effect of phosphorylation on the reporter molecule, whose activity has been rendered sensitive to phosphorylation is measured. Phosphorylation may decrease the activity (i.e. the bioluminescent generating properties) of the reporter molecule and any modulator which inhibits or reduces the activity of the kinase will lead to an increase in the activity (i.e. the bioluminescent generating properties) of the reporter molecule and any modulator which increases the activity of the kinase will lead to an reduction in the activity (i.e. the bioluminescent generating properties) of the reporter molecule.

For the most part, the molecules being sought will be inhibitors of kinase function and in this aspect of the invention there is the advantage that the assay is a positive assay in that the activity of the reporter molecule is increased by the action of the inhibitor. This reduces the possibility of false positive results as opposed to a negative assay in which protein kinase inhibitors could possibly inhibit luciferase activity independent of effects on kinases. With a positive assay false positive results would come from substances that increase transcription and translation of luciferase or increase its stability. Both these processes have different kinetics compared to kinase activities and would therefore be distinguishable from kinase inhibition.

The method is directed to the detection of modulators of kinase function which could be drug candidates. As discussed above, it is an advantage of carrying out the method in vivo that you can determine whether a compound might be suitable for use in vivo. As such, the method of administering the test sample to the transgenic non-human animal can be any method of administration that is suitable for drug administration.

The test sample can be administered orally, rectally, topically, buccally, by inhalation or parenterally (e.g. intramuscularly, subcutaneously, intraperitoneally or intravenously) in the form of an injection or infusion. The preferred administration forms will be administered orally and by injection or infusion. The most preferred administration form will be suitable for oral administration.

For all administration forms, the test sample may be administered in formulations usually containing well-known pharmaceutically acceptable carriers, adjuvants and vehicles. Thus, the test sample may be incorporated, with one or more conventional carriers, diluents and/or excipients, to produce conventional galenic preparations such as tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, sterile packaged powders, and the like. Biodegradable polymers (such as polyesters, polyanhydrides, polylactic acid, or polyglycolic acid) may also be used for solid implants. The compositions may be stabilized by use of freeze-drying, undercooling or Permazyme.

Suitable excipients, carriers or diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, aglinates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, water, water/ethanol, water/glycol, water/polyethylene, glycol, propylene glycol, methyl cellulose, methylhydroxybenzoates, propyl hydroxybenzoates, talc, magnesium stearate, mineral oil or fatty substances such as hard fat or suitable mixtures thereof. The compositions may additionally include lubricating agents, wetting agents, emulsifying agents, suspending agents, preserving agents, sweetening agents, flavouring agents, adsorption enhancers, e.g. for nasal delivery (bile salts, lecithins, surfactants, fatty acids, chelators) and the like. The compositions may be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration by employing procedures well known in the art.

The "test sample" administered to the transgenic non-human animal maybe any sample, for example any sample consisting of or containing the test kinase modulator substance (i.e. the kinase modulator to be tested), e.g. a pure sample, containing only the kinase modulator to be tested or may represent a pool of pure samples or a chemical library e.g. prepared by combinatorial chemistry. The sample may comprise known and/or uncharacterised components. If a sample comprising uncharacterised compounds is found to contain a kinase modulator, then the sample may be fractionated using standard techniques known in the art such as chromatography, (e.g. HPLC, thin-layer chromatography, FPLC, gel filtration, desalting etc.) and the resulting fractions or isolates may then serve as test samples in this assay. In this way it is possible to screen large pools of samples with relatively few assays, and additionally to screen samples that contain new or uncharacterised components without first purifying the components. It is also possible to administer pure samples to the transgenic non-human animal. Thus the test sample may be any sample of pure or impure material, provided in any convenient way e.g. it may be a test substance itself or it may be a composition containing a test substance (which test substance itself may be pure or impure) and a carrier or diluent e.g. an appropriate medium. It may be a crude preparation or a purified or partially purified preparation.

The test sample may comprise synthetic or naturally occurring components. Naturally occurring components may for example be secreted by microorganisms such as bacteria or fungi, and provide a great range of chemical diversity. The test substance may thus be any substance. It may thus be of any chemical nature including both complex and simple molecules, e.g. organic or inorganic molecules. The present invention also relates to modulators of protein kinases identified by the assay method of the invention, particularly protein kinase inhibitors and their use in medicine. All of the prior art documents referred to are incorporated herein by reference. The invention will now be described in more detail in the following non- limiting Examples with reference to the drawings in which:

Figure 1 shows a schematic outline of PBCA signalling and the action of PKA modulators.

Figure 2 shows the luminescence (y axis, in RLU) measured in 293T cells transfected with pGL3 -PKA-luc after incubation with medium with or without a) lOOμM forskolin for 15 minutes b) 500 μm 8-CPT-cAMP. Results are presented as the results of luminometry on cell lysate, as means of 6 replicated to ± SD.

Figure 3 shows the luminescence measured in HepG2 cells transfected with pGL3 -PKA-luc after incubation with medium with or without 1 OμM forskolin for 15 minutes.

Figure 4 shows the luminescence measured in primary mouse skin fibroblasts and COS-I cells transfected with pGL3 -PKA-luc after incubation with medium with or without different concentrations of forskolin for 30 minutes. Luminescence was measured by imaging with IVIS 1004 minutes after addition of luciferin (a) or by luminometry on cell lysates (b).

Figure 5 shows the luminescence measured in primary mouse skin fibroblasts, a) Shows a comparison between the use of the SV40 and CMV promoters to drive PKA-luc expression, b) Shows that under the control of the CMV promoter, PKA-luc remains sensitive to forskolin. Luminescence is measured by imaging.

Figure 6 shows the effect of PKA inhibitor H89 on the response of PKA- luciferase to forskolin. In the presence of forskolin alone, luminescence is reduced. This is reversed by addition of PKA inhibitor H89 for forskolin concentration of lμM and lOμM. For lOOμM forskolin, H89 partially restores luminescence to control levels. Figure 7 shows the effect of treatment with various fruit extracts on PKA luciferase. A) HepG2 cells in 24 well plates were transfected with pCDNA3~ luciferase PKA 24 hours prior to treatment with the indicated extracts made from 30 mg fresh weight pr. ml cell culture medium for 20 hrs. Luciferin was then added to the medium and cells were imaged with IVISlOO. Data represents luminescence from an average of triplicates in a typical experiment ± SD. B) HepG2 cells in 24 well plates were transfected with pCDNA3-luciferasePKA the day before treatment with the indicated concentrations of pomegranate extract. The cells were treated for 24 hours before incubation with luciferin for 4 min and subsequent imaging with IVISlOO. Results are presented as mean of 3 replicates in a typical experiment ± SD. Luminescence increases with the concentration of kinase inhibitor containing extract.

Figure 8 shows the effect of anthocyanidins on LuciferasePKA. A) HepG2 cells in 24 well plates were transfected with ρCDNA3-luciferasePKA and after 20 h treated with the indicated anthocyanidins for 24 hours. Luciferin was added and luminescence was measured after 4 min by imaging with IVISlOO. Results are presented as flux (photons/sec) in an average of 3 replicates +/- SD in a typical experiment. B) HepG2 cells in 24 well plates were transfected with pCDNA3- luciferasePKA the day before incubation for 20 hours with the indicated concentrations of delphinidin (all samples adjusted to 0.1 % of the vehicle DMSO). Luciferin was then added and luminescence was measured after 4 min incubation by imaging with IVISlOO. Luminescence is presented as flux (photons/sec) integrated over 60 sec. Each point is an average of three independent samples ± SD.

Figure 9 Shows the reversal of PKA inhibition by forskolin. HepG2 cells in 24 well plates were transfected with pCDNA3-luciferasePKA the day before treatment as indicated. After 20 h incubation with extract or delphinidin the cells were treated with forskolin (black bars) or vehicle (control, white bars) for 30 min at 37oC. Luciferin was added and luminescence measured by imaging with IVISlOO. Luminescence (average of triplicates in a typical experiment) is presented as % of control (vehicle) ± SD.

Figure 10 shows the effect of pomegranate extract and delphinidin on PKA mediated phosphorylation of kemptide and luciferasePKA activity. HepG2 cells were treated with or without the indicated concentration of pomegranate extract or delphinidin for 28 hours before luciferin was added and luminescence was measured by imaging with (black bars). Cells were then harvested and lysed in PKAassay lysis buffer and aliquots of each lysate were then submitted to measurements of PKA-mediated phosphorylation of kemptide as described in materials and methods (white bars). Luminescence results (black bars) are presented as average of % of control (no treatment) of 3 replicates ± SD. PKA-activity is presented as average of 3 replicates measured in triplicates ± SD.

Figure 11 shows the results of imaging of transgenic pCMV-PKA-luciferase mice (PK mice). (A) Different offspring from mice made pregnant with pronuclear injected eggs were anaesthetized and luciferin was injected i.p. Animals were then imaged with IVIS 100^® Imaging System after 7 min lying on their dorsal side. Two exposures were made, one regular photograph and one in complete darkness. The two images were then superimposed on each other and luminescence is represented by pseudo colouring. The correlation between photon counts and colour is shown in the colour bar. (B) PK8 offspring (littermates, n=5,) were sacrificed by cervical dislocation before indicated tissues and organs were dissected out during 10 minutes. Tissues were homogenized and samples were analyzed by luminometry and data are presented as average of luminescence units, ±SD, on a log scale Y axis. Figure 12 shows in vivo imaging of PKA-luciferase activity and the effect of isoproterenol. A) Imaging of luminescence from two transgenic PKA-luciferase mice. Luciferin was injected i.p. and the mice were imaged after 7 min (0 min). Isoproterenol was then injected (60 μg i.v.) and the mice were imaged with IVIS 100^® Imaging System after 30 min. B) Nine transgenic animals and 5 control animals were injected with luciferin and isoproterenol imaged as above except that pictures were taken at the indicated interval. Luminescence was than quantitated in the abdominal area using the Living Image software.

Figure 13 shows (a) Offspring from PK2 and PK8 were injected with luciferin and imaged after 7 min. Then isoproterenol (60 μg, PK2 (■), n=3, and PK8 (A), n=3) or PBS (300 μl, PK2 and PK8 (♦), n=4) was injected and animals were imaged at the indicated time points. Luminescence is expressed as mean percent of control (before isoproterenol injection), ±SD. (b) Offspring from PK8 were anaesthetized and had the fur over the skull removed before injection of luciferin i.p. The dorsal side of the head was then imaged after 7 min before injection of 60 μg isoproterenol i.v. and subsequent imaging after the indicated time points. Data is presented as percent luminescence of control (before isoproterenol injection), average of three animals, ±SD. The lower panel shows images from a typical experiment, (c) PK8 offspring were anaesthetized with isoflurane and luciferin was injected i.p. After 7 min the hind limb muscles (gastrocnemius) were imaged (basal activity) before 10 μg isoproterenol was administered i.m. (gastrocnemius) at time zero. The animals were then imaged 90 or 210 sec after isoproterenol injection. Luminescence in the muscles were quantitated and presented as mean percent luminescence (compared to basal activity) in both hind limbs, n=4, ±SD. The lower panel shows images from a typical experiment.

Figure 14 shows in vivo imaging of differential response to isoproterenol. Two areas in the abdominal region of PKl 1-1 (indicated by circles) were analysed (A) and quantitated with Living Image (B) before and 30 minutes after isoproterenol injection (60 μg i.v.).

Figure 15 shows in vivo imaging of luciferasePKA in exposed tissues in response to isoproterenol. PK8 offspring was anaesthetized with isoflurane and luciferin was injected i.p. After 7 min the abdomen was opened and several organs and tissues were exposed, the animals were imaged (basal activity) before 60 μg isoproterenol was administered i.v. at time zero. The animal was then imaged 150 sec after isoproterenol injection (left panel). For quantification of response, PK.8 offspring were treated as above and luminescence in the regions marked with circles in the left panel corresponding to the indicated tissues in was quantitated and presented as average percent luminescence of control (before isoproterenol), n=5, ±SD. EXAMPLES

Materials and Methods

Generation ofplasmids:

PKA-luciferase expressed from SV40 promoter (pGL3 -PKA-luc): Luciferase from Photinus pyralis was genetically modified to code for the amino acid sequence

instead of the wild type sequence V₂₁₇R₂₁8F-2i9S220- The modified luciferase (PKA-luc) was obtained by the following procedure:

Luciferase was amplified with PCR primers 5'-CGAAACAAAACAAACTA-3' (SEQ ID NO:35)and S'-GGGGCATGCGAGAATCTCCTGCAGGCAGTTCTATG- 3' (SEQ ID NO:36) with pTAL-luc (BD Biosciences) as template. The PCR product was digested with BgIII and Sphl and ligated into the same sites in pTAL-luc (BD Biosciences). This construct was digested with Ncol and Xbal and the fragment was ligated into pGL3-control (Promega) digested with the same enzymes to obtain pGL3 -PKA-luciferase expressed from the SV40 promoter.

PKA-luciferase expressed from CMV promoter (pCDNA3 -PKA-luc): pGL3-PKA-luc and pCDNA3 (Invitrogen) were digested with HindIII and

Xbal. The 1655 bp fragment from pGL3-PKA-luc was ligated into ρCDNA3 using T4 DNA ligase (Promega) to obtain ρCDNA3 -PKA-luc.

pCMVp-PKA-luciferase expressed from CMVinfront ofintron; It has been shown that in vivo expression of foreign genes is enhanced when the transgene contains an intron between the promoter and the exon. Therefore ρGL3 -PKA-luc was digested with Xbal and HindIII and the resulting fragment was ligated into the Notl site of pCMVβ (Clontech) after treatment with T4 DNA polymerase to achieve blunt end ligation, the obtained construct was termed pCMVp-PKA-luc. PKC-luciferase expressed from CMV promoter (pCDNAS-PKC-luc): The amino acid sequence of PKA-luc (Ra₁₇RFSHARD₂₂₄) was changed to R₂I₇RFSLRRD₂₂₄ by synthetic gene synthesis (Genescript inc. Piscataway, NJ, USA).

Cell culture

Generation of primary mouse fibroblasts

Two mouse ears were washed in 70 % ethanol and then in Hank's Balanced Salt Solution (HBSS, Gibco). The ears were cut into as small pieces as possible with scalpels and transferred to new HBSS. Cells and pieces of tissue were pelleted at 1 g, the supernatant was removed and the pellet washed with HBSS. Pelleted material was then transferred to 75 cm² tissue culture flask and HBSS replaced with 2 ml DM EM (Gibco) with 50% foetal calf serum and Glutamax (Gibco). The cells were then cultured for 2-3 weeks before being aliquoted and frozen in liquid nitrogen.

HepG2 cell culture and transfection

HepG2 cells (passage 80 -110) were routinely cultured in Dulbeccos Modified Eagles Medium (Gibco,Carlsbad, CA, USA) with 10 % fetal calf serum and penicillin/streptomycin with or without the indicated additions. Cells were cultured in 12 or 24 well plates and transfections with pCDNA3-PKAluc were performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) essentially as described by the manufacturer. Briefly, 0.8 - 1.6 μg DNA and Lipofectamin 2000 were diluted separately in OptiMem (Gibco, Carlsbad, CA, USA) and mixed after 10-15 min incubation. After additional 15 minutes the mixture was added to cells cultured in DMEM with 10 % FCS without antibiotics. The transfection mix was left in the cell medium for approx. 20 hours before treatment of cells with PKA modulators or extracts.

Luminescence measurements:

After treatment of cells with extracts or pure substances 100 μg luciferin was added to each well (100 μg/ml final concentration). Cells were incubated for 4 min before imaging for 1 min with IVIS 100 Imaging system (Xenogen, Alameda, CA, USA). Photons emitted per second (flux) was calculated with the software Living Image (Xenogen, Alameda, CA, USA)

Compounds and fruit extracts:

Cyanidin 3-glucoside, delphinidin 3-glucoside, pelargonidin 3-glucoside, delphinidin, pelargonidin, cyanidin and petunidin were obtained from Polyphenols AS (Sandnes, Norway). Acetonitrile for LC-MS analysis was LiChrosolv grade from Merck (Darmstadt, Germany) and water was of Milli-Q quality (Millipore Corp., Ireland). Formic acid was preanalysis from Prolabo (Paris, France). Forskolin was from Sigma- Aldrich (St. louis, MO)

For the fruit extracts, 1Og of homogenized fresh berries or vegetables was dissolved in 10 ml of methanol, mixed vigorously for 15 sec and treated in a ultrasonic water bath for 20 min at O⁰C. The mixture was centrifuged at 3000 g for 5 min before concentrated under vacuum to a viscous liquid. The concentrated was diluted directly in tissue 3 culture cell medium and sterile filtered before storage at - 7O⁰C.

PKA-assay: Activity of PKA in cell homogenates was measured essentially as described by Kemp et al. ((1997) J. Biol. Chem. 252, 4888-94.). Briefly, cells from each well were harvested in PBS and lysed in the presence of 0.5 % Triton-X 100, 5 mM EDTA, 50 mM NaF, 10 mM Na-ρyroρhosρhate, 1 mM PMSF, 1 mM Navanadate, 100 mM NaCl, 50 mM Tris-HCl, pH 7.4. The homogenate was centrifuged for 15 min at 10 000 rpm, and 10 μl of the supernatant was added to a mixture of ATP, Kemptide and ³²P- γATP. The mixture was incubated for 9 min at 3O⁰C, before application of the reaction mix to pieces of P81 chromatographic paper (Whatman, Brentford, UK) for binding. The paper pieces were washed 5 times in 75 mM phosphoric acid and dried before scintillation counting. Production of transgenic animals:

Transgenic mouse lines were created by pronuclear injection of purified DNA into fertilized eggs, (B6CBA F2) according to standard protocols (Hogan et al., 1994 supra). Animal care was in accordance with national legislation and institutional guidelines.

In vivo imaging:

Mice were anaesthetized with isofluorane, injected with —130 mg/kg luciferin i.p. and subjected to whole body imaging using the IVIS 100^® Imaging

System from Xenogen (Alameda, CA₅ USA). Luminescence from the animals were integrated over a period of 1 min after an initial period of 7 min during which luciferin is entering systemic circulation. Images were analysed using the software Living Image^® from Xenogen.

Example 1 : Measurement of PKA activity by luminescence in vitro.

To establish that the modified luciferase faithfully responded to PKA modulators, various cell types were transfected with pGL3-PKA-luc. An outline of cellular components and the function of experimental reagents is shown in Figure 1. 293T cells were extensively used as these cells express high levels of both subunits of PKA, Cells grown in 12 well plates were incubated with pGL3-PKA- luc and Lipofectamin 2000 (Invitrogen) for 6 - 12 hours. Briefly, 60 - 70 % confluent cells in 12 well plates were incubated with a mixture of Lipofectamin 2000 and 1-2 μg DNA/well according to recommendations by the manufacturer (Invitrogen). After 6-12 hours incubation the cells were treated with various PKA modulators as indicated.

293T cells were transfected with pGL3-PKA-luc and treated with medium with or without 1 OOμM forskolin for 15 min. The cells were then lysed and luminescence in the lysate was measured by luminometry as recommended by the manufacturer of Luciferase Assay (Promega). Total cell protein was measured by the BioRad Protein assay as recommended by the manufacturer (BioRad) and used for normalization of luminescence between samples. Luminescence was reduced in the presence of the PKA stimulator forskolin (Figure 2, in which results are presented as means of 6 replicates ± SD).

The transfected cells were then treated with medium with or without 8- cpt-cAMP for 15 min and analysed as described above. As shown in Figure 2b, addition of the PKA agonist 8 CPT-cAMP led to a reduction in luminescence.

In vivo expression of a protein kinase sensitive luciferase for assessment of systemic effects of kinase modulators will require responses in several cell types. To establish that PKA-luc responded in various cell types, pGL3-PKA-luc was transfected into COS 1 cells, HepG2 cells and primary mouse skin fibroblasts, and subsequently treated with PKA modulators.

HepG2 cells were transfected with pGL3 -PKA-luc as described and treated with forskolin at the indicated concentrations. A reduction in luminescence was seen in the presence of forskolin (Figure 3). HepG2 cells are human hepatoma cells and these results show that PKA-luc responds as expected in this cell line derived from liver.

The effect of different concentrations of forskolin on PKA-luc on primary mouse skin fibroblasts transfected with ρGL3-PKA-luc was then assessed.

Cells in passage #2 after dissection were transfected with pGL3 -PKA-luc and treated with the indicated concentrations of forskolin for 30 min (Figure 4a). Luciferin was then added directly to the cells and the cells were imaged after 4 min with IVIS 100™ and luminescence (photond/sec) was quantified with Livinglmage™ according to instruction by the manufacturer (Xenogen).

The results presented in Figure 4a clearly show that PKA-luc responds to forskolin as expected in these cells.

The effect of forskolin on PKA-luc activity was also tested in COSl cell transfected with pGL3PKAluc.

COSl cells were treated as above and luminescence was measured using luminometry on cell lysates. Values were normalized according to total cellular protein and results are presented as means of 6 replicates ± SD (Figure 4b). COSl cells originate from African green monkey kidney epithelium and it is clear from Figure 4b that the cells express the transfected PKA-luc and that the enzyme is responsive to PKA-activity and PKA-modulators.

The expression of PKA-luc in the above experiments is driven by the SV40 promoter. This promoter has been shown to drive expression of transgenes in vivo. However, the CMV-promoter has also been used successfully and is regarded as being more efficient than SV40. To establish that the CMV promoter drives expression of PKA-luc in a manner that is compatible with effect of assessment of PKA modulators, cells were transfected with pGL3 -PKA-luc or ρCDNA3-PKA- luc.

To compare the effect of the SV40 and CMV promoters in primary mouse fibroblasts, cells were transfected with either pGL3-PKA-luc or pCDN3-PKA-luc. After 12 hours, luminescence was measured by imaging. The results are shown in Figure 5a and are presented as means of 3 replicates ± SD. The results in Figure 5 a demonstrate that the CMV promoter is far more efficient than the SV40 promoter driving expression of PKA-luc. To establish that PKA-luc expression driven by CMV promoter also responded to PKA activity modulation, 293T cells or primary mouse fibroblasts were transfected with pCDNA3-PKA-luc and treated with either Forskolin or H89 or both (results shown only for primary mouse fibroblasts, Figure 5b).

The effect of forskolin on PKA-luc driven by CMV promoter in primary mouse fibroblasts was also tested. Cells were transfected with pCDNA3-PKA-luc and subsequently treated with 100 μM forskolin for 15 min. Luminescence was then measured by imaging. Results are presented in Figure 5b as mean of 3 replicates ± SD. The results in Figure 5b show clearly that PKA activity modulation by forskolin is independent of which promoter that drives luciferase expression.

Example 2 - Effect of inhibitors of PKA The effect the PKA inhibitor H89 (an inhibitor of protein kinase A) on forskolin induced PKA activity was then tested. The effect of forskolin and H89 on luminescence from PKA-luc in primary mouse fibroblasts was compared. Cells were transfected with pCDN A3 -PKA-luc and treated with the indicated concentrations of forskolin (Figure 6A) or forskolin and 10 μM H89 (Figure 6B). Luminescence was then measured by imaging. The results are shown in Figure 6 and are presented as mean of 3 replicates ± SD.

From Figure 6 it can be seen that the effect of forskolin could be reversed by the PKA inhibitor H89 at least at the lower forskolin concentration range.

The effect of crude mixtures of protein kinase inhibitors was also tested. Various plants and plants extracts are known to contain protein kinase inhibitors. We used one such extract to demonstrate that the PKA-luc enzyme can be used for detection of kinase inhibitory activity of unknown origin. HepG2 cells were transfected with pCDN A3 -PKA-luc and treated with a water/methanol extract of pomegranate at various concentrations.

Various food plants suspected to contain protein kinase inhibitory activity was used for production of extracts. HepG2 cells were transfected with pCDNA3- PKA-luc and treated with a water/methanol extract of pomegranate, strawberries, black currants, blueberries, crowberries or raspberries (fig. 7) for 20-24 hours. Of these extracts the pomegranate and strawberry extract gave the highest induction of luminescence (close to 3 fold higher than control, fig 7A), demonstrating that these extracts contain one or several substances which inhibit PKA activity (i.e. reduce phosphorylation of luciferase). Different concentrations of the pomegranate extract were tested (fig. 7B): 30 mg fresh weight pr. ml cell medium was found to give the highest induction with no further increase at 100 mg/ml.

To make the pomegranate homogenate, it was homogenized and extracted with equal volumes of methanol. The extract was lyophilized and dissolved in RPMI-1640 with 2% FCS before being added to HeρG2 cells that had been transfected with pCDN A3 -PKA-luc at the indicated concentrations (mg fresh weight/ml cell culture medium). Cells were incubated for 24 h before addition of luciferin and imaging. Results are presented as mean of 3 replicates ± SD. The pomegranate extract increases luminescence from the cells, suggesting that luminescence from PKA-luc is to some extent inhibited by a basal PKA activity (Figure 7). Furthermore, the results indicate that the extract inhibits this PKA activity. The inhibition of PKA activity can partly be overcome by treatment of cells with forskolin (data not shown).

Pomegranate is exceptionally rich in anthocyanins and the seed coat contains glucosides of delphinidin, cyanidin and pelargonidin. Anthocyanidins (anthocyanin aglycones) are structurally related to flavonoids which have been used extensively as protein kinase inhibitors in experimental systems. We therefore tested several anthocyanidins found in pomegranate with respect to PKA inhibition in HepG2 cells. We found that, of delphinidin, pelargonidin, petunidin and peonidin, delphinidin was the only compound giving significant inhibition of basal PKA activity in HepG2 cells, fig. 8A. Delphinidin 3-glucoside at the same concentrations was less effective (data not shown). Interestingly, the only difference between petunidin and delphinidin is the presence of a methoxy group in position 3' in the B- ring in petunidin instead of a hydroxyl group as in delphinidin. Luminescence was found to respond to delphinidin in a dose dependent manner (fig. 8B) and the effect was found to increase over time with a maximum at approximately 24 hrs after addition to the cells (data not shown).

The increase in luminescence seen with extracts from the cells without previous stimulation of PKA suggests that at least in HeρG2 cells luminescence from luciferasePKA is to some extent inhibited by a basal PKA activity. To test whether extract mediated PKA inhibition could be overcome by stimulation with forskolin cells were treated with delphinidin or pomegranate extract with or without subsequent treatment with forskolin. Fig. 9 shows that both extract and delphinidin inhibition of PKA can be partly overcome by treatment of cells with the PKA activating drug forskolin. It could be argued that the observed effects of pomegranate extract and delphinidin are directly linked to the enzymatic oxidation of luciferin to oxyluciferin mediated by luciferase and ATP. Certain polyphenols have been shown to bind to ATP -binding pockets and one could imagine that polyphenols in the extract could bind to the ATPbinding pocket of luciferase. This would, however, most likely inhibit luciferase activity and decrease luminescence and not the opposite as observed in this study. To provide evidence that the increase in luminescence is linked to intracellular PKA activity the effect of pomegranate extract and delphinidin was measured by a conventional assay in which PKA incorporates ³²P into a synthetic peptide containing a PKA target sequence (kemptide) with subsequent scintillation counting of radioactivity. Cells were transfected with pCDNA3-Luc and treated with extract or delphinidin as indicated and luminescence was measured by imaging. After imaging the same cells were lysed and cell homogenates were assessed for PKA activity. Fig. 10 clearly demonstrates that delphinidin and pomegranate extracts inhibit PKA activity as assessed by both the increase in luminescence as well as the decrease in phosphorylation of kemptide.

Example 3 - In vivo imaging of protein kinase activity

Based on the cell culture experiments we produced transgenic mice with PKA-luciferase driven by the CMV promoter. In the construct used for animal production we also included an intron sequence which has been shown to improve expression of foreign genes in vivo. Of 90 injections, 33 offspring were obtained in 9 litters. The offspring were tested for expression of PKA-luciferase by injection of luciferin and subsequent imaging with an ultra sensitive video camera (IVIS 100). Examples of animals with highest activities are shown in Figure 11a.

The difference among founders may be explained by insertion of the transgene in the genome at different loci with different transcriptional activity.

However, there seems to be a recurrent pattern, in that all 8 animals have one or two bright spots in the abdominal region and 4 animals have strong luminescence also from the thoracic region (PK7,11-1, 12 and 14). Disregarding paws and the nose region most positive animals displayed the highest reporter activity in the abdominal region, but there were considerable differences in the luminescence pattern in this region. There was apparently no correlation between luminescence pattern and sex.

The CMV promoter is regarded as a strong constitutive promoter and yields high expression in many cells transfected with CMV containing expression vectors.

We have also found that the CMV promoter drives expression of PKA-luciferase in several cell lines (data not shown). The differences in bioluminescence from various organs were substantiated by bioluminescence measurement in tissue homogenates from one of the founders, PK8 (Fig. 1 Ib). The largest difference was between liver and muscle (approx. 200 fold). The individual response varied significantly, particularly in the liver and in the stomach. A possible explanation is that individual mice were in metabolically different status regarding stress and/or feeding, conditions known to alter PKA activity. Another possible explanation for the diversity in expression among tissues/organs is that expression of the transgene takes place in all tissues but basal protein kinase activity is high in most tissues and would thus inhibit luciferase, whereas in other tissues PKA activity is low. One complicating factor is that the CMV promoter has 4 CRE recognition sites. Thus, high PKA activity should yield efficient transcription of PKA-luciferase and strong luminescence. On this ground one could speculate that the luminescence seen in the animals is lower than expected due to low PKA -mediated transcription of the reporter. In long term experiments with PKA-modulators, both enhancers and inhibitors, this could give rise to an underestimation of effects. It should be noted though that PKA modulated CMV transcription in vivo has yet to be demonstrated. To test if PKA-luciferase responded to fast PKA-modulation by beta- adrenergic agonists, all positive founder and 5 control animals were submitted to injection with 60 μg isoproterenol i.v. after first being anaesthetized and injected with luciferin i.p. The animals were then imaged after 30 min. Particularly the founder PK 8 and 12 displayed a dramatic reduction in luminescence, luminescence in the abdominal region was reduced to approximately 10 % after 30 min (Figure 12A). We then tested all positive founders for response to isoproterenol. Photons from defined areas in the abdominal region were then counted at various time points after injection while the mice were still anaesthetised. Flux (photons/sec) was then related to luminescence at time 0 (set to 100 %). It is clear from Figure 12B that luminescence from the abdominal region is reduced significantly after 5 min in several animals (PK2, 8, and 12), whereas luminescence was modestly increased in control animals. It should be noted that the luminescence from all animals, including the controls, is slightly reduced from 20 to 30 min after isoproterenol injection. This is probably due to reduced availability of luciferin or inhibition of the luciferase by oxyluciferin, the end product of the reaction.

Two founders responded poorly (PKl 1 and PKl 7), possibly due to expression of the luciferase in β-adrenergic non-responsive tissue. Imaging of abdominal regions at time points between 0 and 6 min after isoproterenol injection shows that a rapid initial response (1 min) is partly reversed over the next 5 minutes as compared to the control, suggesting that phosphorylation of luciferasePKA is reversible (Fig. 13a). This reversal may be mediated by phosphatase Pl (PPl) and reflect a physiologically relevant response to a strong β-adrenergic stimulus in several organs, although translation of unphosphorylated and enzymatically active luciferase can not be ruled out. β-adrenergic responses are particularly interesting in brain and muscles, as PKA mediate aminotropic and metabolic responses in the two tissues, respectively. We therefore performed in vivo imaging of these tissues in live animals after injection of isoproterenol and found substantial decrease in bioluminescence (i.e. increased PKA activity, Fig. 13b and c). Interestingly, bioluminescence observed from the outside of the brain was not evenly distributed, a bright spot was observed at the position where fissure longitudinalis cerebri ends against the cerebellum. This could possibly reflect variability in PKA activity in different brain structures, although differences in expression can not be ruled out and needs further testing. PKA plays a central role in several brain functions including long term potentiation and memory. Our results demonstrating in vivo measurements of brain PKA activity suggests that this model may be very useful for studies of PKA in the brain as drug target. Close inspection of some of the animals, for example PKl 1-1, shows that luminescence goes down only in some region and not in others, Figure 14A and 14B. In PKl 1-1 luminescence in a bright spot on the upper ventral abdomen (area 1) is virtually unaffected whereas it is dramatically reduced in a lower part of the abdominal region (area 2). One possible explanation is that tissues in area 1 do not express beta-adrenergic receptors.

Inspection of whole body images revealed several bright spots in the abdominal region that displayed reduced bioluminescence after isoproterenol injections, whereas skin in the extremities and nose region responded less clearly to the β-adrenergic stimulus. To quantify this variation and identify some of these tissues/organs, we injected luciferin into PK8 mice before tissues and organs were exposed while the animal was still alive. Isoproterenol was then injected i.v. and images were taken 150 sec after injection (Fig. 15). Photons from selected identifiable organs and tissues were counted and related to luminescence before isoproterenol injection (Fig. 15b). The liver responded to the largest extent and luminescence was reduced to 40 % of control, whereas fat responded poorly (75% of control).

Example 4 - Other protein kinase-sensitive luciferases.

The target sequence for protein kinase C has been introduced into the same area of luciferase as the PKA sequence. This can be tested for responses to PKC modulators.

Claims

1. A transgenic non-human animal which expresses a bioluminescent signal- generating reporter molecule which has been modified relative to the wild type, or unmodified, reporter molecule to introduce a phosphorylation site such that generation of the bioluminescent signal by the reporter molecule is sensitive to phosphorylation at said phosphorylation site.

2. The transgenic non-human animal of claim 1 wherein said reporter molecule is an enzyme which catalyses a chemical reaction which produces light

3. The transgenic non-human animal of claim 1 or 2 wherein the reporter molecule is a luciferase.

4. The transgenic non-human animal of any one of claims 1 to 3 wherein phosphorylation of the modified reporter at the introduced phosphorylation site induces the bioluminescent signal to be generated.

5. The transgenic non-human animal of any one of claims 1 to 3 wherein phosphorylation of the modified reporter at the introduced phosphorylation site reduces production of the bioluminescent signal.

6. The transgenic non-human animal of any one of claims 1 to 5 which is a rodent.

7. The transgenic non-human animal of any one of claims 3 to 6 wherein the luciferase into which the phosphorylation site is introduced is a wild type luciferase.

8. The transgenic non-human animal of any one of claims 3 to 6 wherein the luciferase into which the phosphorylation site is introduced is a mutant luciferase.

9. The transgenic non-human animal of any one of claims 3 to 8 wherein the luciferase into which the phosphorylation site is introduced is a click beetle or firefly luciferase, or mutant thereof.

10. The transgenic non-human animal of any one of claims 3 to 8 wherein the luciferase into which the phosphorylation site is introduced is a Photinus pyralis luciferase, or mutant thereof.

11. The transgenic non-human animal of any one of claims 1 to 10 wherein the introduction of the phosphorylation site is caused by substituting, adding or removing 10 or less amino acids.

12. The transgenic non-human animal of any one of claims 1 to 11 wherein the phosphorylation site that is introduced is a site for a tyrosine kinase.

13. The transgenic non-human animal of claim 12, wherein the introduced phosphorylation site is (K/R) X X (D/E) X X X Y or K/R X X X (DfE) X X Y, where X is any amino acid.

14. The transgenic non-human animal of any one of claims 1 to 11 wherein the phosphorylation site that is introduced is a site for a serine or threonine kinase.

15. The transgenic non-human animal of any one of claims 1 to 14 wherein the phosphorylation site that is introduced is selected from (i) S(P)-X-X-SZT;

(ii) S/T-X-X-E;

(iii) S-X-X-X-S(P);

(iv) S/T-P-X-R/K;

(v) R-X-X-S/T; (vi) R-X-X-S/T-V;

(vii) P-X-S/T-P;

(viii) X-X-S/T-P; (ix) T-X₆-Y;

(x) S-E-G;

(xi) S-X₃-A-X₄-X₅;

(xii) R/L-R/L-X-S/T; (xiii) R/L-R/L-X_rS/T-X₂; and

(xiv) T/S-X-R/K

wherein S(P) indicates a phosphorylated serine residue, X is any amino acid, X₁ is any amino acid, X₂ is a hydrophobic amino acid, X₃ is M, V, I or K, X₄ is N or K, X₅ is S or T and X₆ is E, P, D or G.

16. The transgenic non-human animal of claim 15 wherein Xi is a small amino acid.

17. The transgenic non-human animal of claim 15 wherein the introduced phosphorylation site is R R X S.

18. The transgenic non-human animal of claim 14, wherein the introduced phosphorylation site is TXR.

19. The transgenic non-human animal of any one of claims 3 to 20 wherein the introduced phosphorylation site is in a hinge region between two functional sites or in a functional domain.

20. The transgenic non-human animal of claim 19 wherein the introduced phosphorylation site is in the flexible loop of P. pyralis luciferase (amino acids 436- 440) or the equivalent residues in another luciferase molecule.

21. The transgenic non-human animal of claim 19 wherein the introduced phosphorylation site is in the luciferin binding site or in the ATP binding domain, preferably in an α helix comprising part of the ATP binding domain.

22. The transgenic non-human animal of claim 21 wherein the introduced phosphorylation site is in residues 217-220 or 217-224 of the P pyralis firefly luciferase sequence, or the equivalent residues in another luciferase molecule.

23. The transgenic non-human animal of claim 21 or 22 wherein said reporter molecule is luciferase having the sequence RRXS, preferably RRFS at residues 217- 220 of the Photinus pyralis luciferase sequence, or the equivalent residues in other luciferase molecules.

24. The transgenic non-human animal of any one of claims 21 -23 wherein said reporter molecule is luciferase having the sequence RRFSLRRD at residues 217-224 of the Photinus pyralis luciferase sequence, or the equivalent residues in other luciferase molecules.

25. The transgenic non-human animal of any one of claims 3 to 11 wherein the introduced phosphorylation site is in the C terminal domain of luciferase, preferably between residues 537-550 of the P. pyralis firefly luciferase or the equivalent residues in another luciferase molecule.

26. A method of making a transgenic non-human animal of any one of claims 1 to 25, said method comprising the step of introducing into said animal a nucleotide molecule encoding an bioluminescent signal generating reporter molecule, which has been modified relative to the wild type, or unmodified reporter molecule to introduce a phosphorylation site such that the bioluminescent signal generating properties of the reporter molecule are sensitive to phosphorylation at the said phosphorylation site.

27. The method of claim 26 comprising the steps of

(a) introducing a recombinant genetic construct encoding an bioluminescent signal generating reporter molecule, which has been modified relative to the wild type, or unmodified reporter molecule to introduce a phosphorylation site such that the bioluminescent signal generating properties of the reporter molecule are sensitive to phosphorylation at the said phosphorylation site under the control of a promoter into the pronucleus of a fertilised egg, and

(b) implanting said egg into a psuedopregnant foster mother.

28. The method of claim 26 comprising the steps of

(a) introducing a recombinant genetic construct encoding a bioluminescent signal generating reporter molecule, which has been modified relative to the wild type, or unmodified reporter molecule to introduce a phosphorylation site such that the bioluminescent signal generating properties of the reporter molecule are sensitive to phosphorylation at the said phosphorylation site under the control of a promoter into an ES cell,

(b) introducing said ES cell into a blastocyst and implanting said blastocyst into a pseudopregnant foster mother.

29. A method for identifying the presence of a modulator of protein kinase function in a test sample said method comprising the steps of: a) administering the test sample to a transgenic non-human animal as defined in any one of claims 1 to 25, and b) assessing the modulation of protein kinase function by comparing the behaviour of the reporter molecule in the presence and absence of said test sample.

30. The method of claim 29 wherein the activity of the reporter molecule is assayed non-invasively in vivo.

31. The method of claim 29 or 30 wherein the activity of the reporter molecule is assayed using an in vivo imaging system based upon detection of the bioluminescent signal from the reporter.

32. The method of any one of claims 29 to 31 wherein the modulator of protein kinase function is a specific activator or inhibitor of a protein kinase.

33. The method of any one of claims 29 to 32 wherein said modulator is an inhibitor of a protein kinase.

34. The method of any one of claims 29 to 33 wherein said test sample is a pure sample or a pool of pure samples.

35. A modulator of a protein kinase identified by the assay method of any one of claims 29 to 34.

36. The modulator a protein kinase of claim 35 for use in medicine.