WO2002090992A2

WO2002090992A2 - Screening method

Info

Publication number: WO2002090992A2
Application number: PCT/GB2002/001946
Authority: WO
Inventors: Peter Andrews; Jon Draper; James Walsh
Original assignee: Axordia Ltd.
Priority date: 2001-05-04
Filing date: 2002-04-29
Publication date: 2002-11-14
Also published as: GB0111004D0; AU2002249467A1; WO2002090992A3

Abstract

A screening method is disclosed which enables the identification of biologically active agents which mediate their effects through the activation of genes. The method utilises promoter trap, enhancer trap and gene trap constructs, comprising a reporter molecule, which are transfected into cells which are then cloned into a cell array. The cell array is exposed to an agent(s) to be tested and the response of the cell array to the agent monitored. The method enables both the identification of agents and the genes through which the agents act.

Description

SCREENING METHOD

The invention relates to a screening method for the identification of biologically active agents; agents identified by the method; and genes involved in mediating responses to biologically active agents.

The discovery of biologically active agents is a multiple step process which involves the screening of many thousands of potential compounds for their activity against cellular targets. The methodology typically involves the following steps: i) the development of an assay which facilitates the identification of compounds with the desired activity; ii) validation of the assay; iii) optimisation of the assay to provide an assay which allows the reliable detection of the effects of an agent; and iv) the collation and representation of data generated by the assay.

Developments in molecular genetics and cell biology provide the basis for rational drug screening and design. In particular, understanding the mechanisms that regulate cell behaviour provides opportunities to identify agents that will modulate cell function. One approach is to identify particular proteins, or other molecules, that play specific roles in signalling pathways. Such regulatory molecules provide the targets for drug design and screening. Clearly, a drawback of this approach is that the targets must first be identified.

An alternative is to develop assays that relate to specific patterns of cell behaviour without necessarily knowing the identity of the specific molecular target. For example, by gene array technology it is possible to assess the patterns of expression of a large number of genes in response to a range of test compounds in comparison with the pattern observed by exposure to a specific reference compound. Compounds that induce the same pattern of gene activity are likely to interact with the same signalling pathway, though not necessarily with the same target molecule. One problem with this approach is that only a snapshot at a particular point in time can be assessed. Another is that many changes in gene activity assessed during array experiments are relatively small, less than 2 or 3 fold changes in expression, so that meaningful analysis depends upon replicate assays with sophisticated statistical tools.

An alternative approach could be based upon a 'Cell Array' of different cloned derivatives of a particular cell type in which a reporter gene (for example Green Fluorescent Protein (GFP)) is expressed under the control of a range of different gene promoters. For example, cell lines would be transfected with a gene trap vector encoding a reporter. In this way a 'library' of independent clones in which reporter expression is regulated by different endogenous regulatory elements could be obtained.

This approach might be applied to a range of cell types, for example, and not by way of limitation, pathogenic bacterial species in the search for new antibiotics and antiseptic agents. Currently methods to control microbial organisms include the use of antimicrobial agents (antibiotics) and disinfectants. These have proved to be problematic since exposure to these agents place a significant selection pressure resulting in the creation of resistant microbes which can avoid the effects of the antimicrobial agent(s).

It is estimated that there are up to 50 million people world- wide infected with drug resistant tuberculosis (TB) (Figures from the World Health Organisation, 1998). In the past the use of antibiotics to treat TB relied on the administration of single drugs (eg ethionamide) which promoted a relatively high frequency of resistance. For this reason, combinations of drugs are now used to treat tuberculosis.

A further example of a pathogenic organism which has developed resistance to antibiotics is Staphylococcus aureus. This is a particular problem in hospitals where patients may have surgical procedures and/or be taking immunosuppressive drags. These patients are much more vulnerable to infection with S.aureus because of the treatment they have received. Resistant strains of S.aureus have arisen in recent years. Methicillin resistant strains are prevalent and many of these resistant strains are also resistant to several other antibiotics.

Chemotherapeutic agents active against pathogenic parasitic species may also be identified by such screening methods. Many parasites have developed elaborate means to avoid immune detection. For example, Trypanosoma brucei spp, the causative agent of African sleeping sickness, has evolved very effective means to evade detection by a host's immune system by periodically switching the dominant cell surface antigen. A large repertoire of over 100 cell surface antigens can be drawn on by the parasite during infection, hi other examples, (eg Plasmodium spp) the parasite replicates within a cell and is therefore shielded from the host's immune system. There is therefore a need to identify chemotherapeutic agents effective at controlling such parasitic diseases.

A large number of primary cell lines and established transformed cell lines are now available derived from tissues of many multicellular organisms, but most notably from mammals such as mice and humans. These cell lines may often retain differentiated functions pertinent to their tissue of origin and many have been used to screen for compounds that interact with some biological activity expressed in such tissues. Cancer derived cell lines have been particularly used to identify compounds that interfere with the proliferation or other functions of such cancer cells. However, cancer cell lines often retain some degree of the biological functions typical of their tissue of origin, and may be used as a source of potential drug targets pertinent to such tissues.

A further cell-type to which the screening method could be applied are embryonic stem cells. During mammalian development those cells that form part of the embryo up until the formation of the blastocyst are said to be totipotent (e.g. each cell has the developmental potential to form a complete embryo and all the cells required to support the growth and development of said embryo). During the formation of the blastocyst, the cells that comprise the inner cell mass are said to be pluripotential (e.g. each cell has the developmental potential to form a variety of tissues). These cells are referred to as embryonic stem cells.

Embryonic stem cells, may be principally derived from two embryonic sources. Cells isolated from the inner cell mass are termed ES cells, hi the laboratory mouse, similar cells can be derived from the culture of primordial germ cells isolated from the mesenteries or genital ridges of days 8.5-12.5 post coitum embryos. These would ultimately differentiate into germ cells and are referred to as embryonic germ cells, EG cells. Each of these types of pluripotential cell has a similar developmental potential with respect to differentiation into alternate cell types, but possible differences in behaviour (eg with respect to imprinting) have led to these cells to be distinguished from one another.

Until very recently, in vitro culture of human ES cells was not possible. The first indication that conditions may be determined which could allow the establishment of human ES cells in culture is described in WO96/22362, the contents of which are incorporated by reference. The application describes cell lines and growth conditions which allow the continuous proliferation of primate ES cells which exhibit a range of characteristics or markers which are associated with stem cells having pluripotent characteristics.

More recently Thomson et al (1998) have published conditions in which human ES cells can be established in culture. The above characteristics shown by primate ES cells are also shown by the human ES cell lines. In addition the human cell lines show high levels of telomerase activity, a characteristic of cells which have the ability to divide continuously in culture in an undifferentiated state. Another group (Reubinoff et. al, 2000) have also reported the derivation of human ES cells from human blastocyts. A third group (Shamblott et. al., 1998) have described human EG cell derivation. As a model system to study ES cell biology we have used embryonal carcinoma cells (EC cells) which are stem cells of teratocarcinomas (e.g. Martin, 1980; Andrews 1988). The stem cells of early embryos and the stem cells of teratocarcinomas have been demonstrated experimentally to be capable of substituting for one another in their respective roles. Thus, embryonal carcinoma cells derived from a spontaneous germ cell carcinoma may participate in embryonic development, and generate normal somatic tissue following injection into a blastocyst (Brinster 1974; Mintz and Illmensee 1975; Papaioannou et al 1975). This clearly demonstrates that murine EC cells may respond to developmental cues in an appropriate manner, and that their differentiation may provide information pertinent to normal embryogenesis. Similarly, human EC cells may provide an insight into the processes that regulate human development (e.g. Andrews 1984; Andrews 1998; Przyborski et al 2000).

Human embryonic stem cells, whether derived from teratocarcinomas (EC cells) or embryos (ES cells), provide the basis for a wide range of screening tests with special relevance to cell differentiation in embryos and adults, . which, in turn may be pertinent to a range of therapeutic targets including tissue regeneration and repair, cancer, viral diseases and embryo toxicology.

If the screen were applied to differentiating EC or ES cells, it would be possible to identify agents active in one specific lineage, but not another. The particular advantage of applying this approach to stem cells such as EC or ES cells, is that multiple assays could be set up in relation to a variety of different cell lineages starting from the same array of reporter transfected clones. Further, such assays would be relevant to the processes that occur during embryogenesis, and so appropriate for the identification of drug candidates that might affect embryonic processes, or pathological events occuring during embryogenesis. Further, EC and ES cells are sources of specific differentiated cell types, often present in adults but otherwise difficult to obtain in large numbers with reproducible properties. Such an approach could be used in a variety of situations. It could be used in screening potential drugs for their ability to activate certain drug targets in a high- throughput assay. It could be used to identify relationships between signalling pathways and specific signals that could be useful in eventually directing the differentiation of ES cells. It could be used in toxicology assays by testing for unwanted activation or inhibition of specific signalling pathways. In this case EC or ES cells are particularly appropriate in screens for embryo toxicology.

The screen is applicable to a set of reporter transfectants of any cell type that might offer particular advantages for a specific search for potential new drags or agents, depending upon the desired target.

According to a first aspect of the invention there is provided a screening method for the identification of biologically active agents comprising: i) providing a population of cells which have been stably transfected/transformed with a nucleic acid molecule encoding a reporter molecule; ii) cloning the transfected cells into a cell array; iii) exposing the array to at least one agent to be tested; and iv) detecting a signal generated by the reporter molecule as a result of exposure to said agent.

In a preferred method of the invention the screen has the further additional steps of i) collating the signal (s) generated by the reporter molecule; ii) converting the collated signal(s) into a data analysable form; and optionally iii) providing an output for the analysed data.

In a further preferred method of the invention the biologically active agent is an antagonist.

In an alternative preferred method of the invention, the biologically active agent is an agonist. The term antagonist is to be construed as any agent capable of inhibiting a biological activity or cellular function, for example the inhibition of a signal transduction pathway; the inhibition of the cell-cycle; the inhibition of transcription; agents which destabilise RNA; the inhibition of translation; the inhibition of post-translational modification to proteins; the inhibition of cell-differentiation; agents which inhibit protein secretion, agents which inhibit cell migration or cell invasion, agents which inhibit cell membrane electrical activity, or agents which inhibit apoptosis.

The term agonist is to be construed as any agent capable of promoting a biological activity or cellular function, for example agents which stimulate signal transduction pathways; agents which promote cell-division; transcription effectors which enhance transcription; translation effectors which enhance translation of polypeptides; agents which enhance the stability of RNA; agents which promote cell differentiation; agents which promote polypeptide secretion; agents which promote cell migration or cell invasion, agents which promote membrane electrical activity, agents which stimulate apoptosis.

hi a yet further preferred method of the invention said cells are eukaryotic cells. Preferably said eukaryotic cells are: protozoan; fungal; insect; plant or mammalian cells.

More preferably still said protozoan is selected from: Plasmodium spp; Plasmodium falciparum; Leishmania spp; Leishmania major; Trypansoma brucei spp; Trypanosoma brucei brucei; Trypanosoma brucei rhodesiensis; Trypanosoma cruzi; Giardia spp.; Cryptosporidium spp. Acanthamoeba spp.; Babesia spp.; Babesia bovis; Toxoplasma spp; Entamoeba spp. Naegleria spp.

In a further preferred method of the invention said species is fungal in origin. More preferably still said fungal species is selected from: Saccharomyces cerevisiae; Candida spp.; Candida albicans. In a further preferred method of the invention said mammalian cells are murine. More preferably still, said mammalian cells are human.

In a fiirther preferred method of the invention said human cells are primary cell lines established from primary cultures of explanted human tissues. Such primary cell lines may include fibroblasts, keratinocytes, endothehal cells, renal tubule cells, neural cells or hepatocytes.

In a further preferred method of the invention said human cells are established cell lines derived either by spontaneous transformation of primary cells, or by infection with a transforming virus, or by transfection with a transforming gene, or derived by the explantation of a tumour.

In a further preferred method of the invention said human cells are stem cells. Preferably said stem cells are selected from the following group: haemopoietic stem cells; neural stem cells; bone stem cells; muscle stem cells; embryonic stem (ES) cells; embryonal germ (EG) cells; mesenchymal stem cells, trophoblastic stem cells, epithelial stem cells (derived from organs such as the skin, gastrointestinal mucosa, kidney, bladder, mammary glands, uterus, prostate and endocrine glands such as the pituitary), endodermal stem cells (derived from organs such as the liver, pancreas, lung and blood vessels).

In a further preferred method of the invention said stem cells are embryonal carcinoma cells. Preferably said embryonal carcinoma cells are TERA2 cells. Ideally said embyonal carcinoma cells are NTERA 2 cells.

The TERA2 cell line was derived from a lung metastasis of a human teratocarcinoma in the mid 1970s (Fogh and Trempe, 1975). Morphologically, TERA2 cultures are quite divergent from the characteristic EC phenotype and display significant heterogeneity, suggesting that these cells undergo spontaneous differentiation (Andrews et al, 1980). However, a tumour containing both embryonal carcinoma cells and differentiated derivatives was produced following injection of TERA2 into a nude mouse host (Andrews et al., 1983a; Andrews et al., 1983b; Andrews et al., 1984). A cell line established from the EC component of this tumour, named NTERA2, closely resembled and maintained the characteristic EC phenotype in culture and, unlike the parent line, was able to produce teratocarcinoma in nude mice with high frequency (Andrews et al., 1983a; Andrews et al., 1983b; Andrews et al.,' 1984). Additionally, various subclones of NTERA2 exhibited the ability to differentiate extensively in vitro following treatment with chemical inducers (such as retinoic acid (RA) or, hexamethylene bisacetamide (HMBA), (Andrews, 1984; Andrews et al., 1986), or with cytokines such as members of the bone morphogenetic protein family (Andrews et al 1994).

In a yet further preferred method of the invention said cell is a prokaryotic cell, preferably a bacterial cell selected from the following group: Staphylococcus aureus;

Staphylococcus epidermidis; Enterococcus faecalis; Mycobacterium tuberculsis;

Mycobacterium bovis; Mycobacterium leprae; Streptococcus group B;

Streptoccocus pneumoniae; Streptoccocus equi; Streptoccocus pyogenes;

Streptoccocus coelicolor; Helicobacter pylori; Neisseria gonorrhea; Streptococcus group A; Borrelia burgdorferi; Coccidiodes immitis; Histoplasma sapsulatum;

Neisseria meningitidis type B; Neisseria meningitidis type C Shigella flexneri;

Escherichia coli; Haemophilus influenzae; Bacteriodes fragilis; Bordetella bronchiseptica; Bordetella parapertussis; Bordetella pertussis; Burkholderia psuedomallei; Campylobacter jejuni; Clostridium difficile; Cornybacterium diphtheria; Salmonella typhi; Yersinia enter ocolitica; Yersinia pestis.

Methods to transfect or transform cells according to the invention are well known in the art. Conventional methods to introduce DNA into cells are well known in the art and typically involve the use of chemical reagents, cationic lipids or physical methods. Chemical methods which facilitate the uptake of DNA by cells include the use of DEAE -Dextran ( Vaheri and Pagano Science 175: p434) . DEAE-dextran is a negatively charged cation which associates and introduces the DNA into cells but which can result in loss of cell viability. Calcium phosphate is also a commonly used chemical agent which when co-precipitated with DNA introduces the DNA into cells (Graham et al Virology (1973) 52: p456).

The use of cationic lipids (eg liposomes ( Feigner (1987) Proc.Natl.Acad.Sci USA, 84:p7413) has become a common method since it does not have the degree of toxicity shown by the above described chemical methods. The cationic head of the lipid associates with the negatively charged nucleic acid backbone of the DNA to be introduced. The lipid/DNA complex associates with the cell membrane and fuses with the cell to introduce the associated DNA into the cell. Liposome mediated DNA transfer has several advantages over existing methods. For example, cells which are recalcitrant to traditional chemical methods are more easily transfected using liposome mediated transfer.

More recently still, physical methods to introduce DNA have become effective means to reproducibly transfect cells. Direct microinjection is one such method which can deliver DNA directly to the nucleus of a cell ( Capecchi (1980) Cell, 22:p479). This allows the analysis of single cell transfectants. So called "biolistic" methods physically shoot DNA into cells and/or organelles using a particle gun ( Neumann (1982) EMBO J, 1: p841). Electroporation is arguably the most popular method to transfect DNA. The method involves the use of a high voltage electrical charge to momentarily permeabilise cell membranes making them permeable to macromolecular complexes. However physical methods to introduce DNA do result in considerable loss of cell viability due to intracellular damage. These methods therefore require extensive optimisation and also require expensive equipment.

More recently still a method termed immunoporation has become a recognised technique for the introduction of nucleic acid into cells, see Bildirici et al, Nature 405, 769. The technique involves the use of beads coated with an antibody to a specific receptor. The transfection mixture includes nucleic acid, typically vector DNA, antibody coated beads and cells expressing a specific cell surface receptor. The coated beads bind the cell surface receptor and when a shear force is applied to the cells the beads are stripped from the cell surface. During bead removal a transient hole is created through which nucleic acid and/or other biological molecules can enter. Transfection efficiency of between 40-50% is achievable depending on the nucleic acid used.

Other, non-liposome based, chemical transfectant agents have become available, for example ExGen500 (polyethylenimine), produced by MBI Feπnentas. ExGen500 is particularly effective for transfection of human ES cells (Eiges, 2001).

The screening of large numbers of agents requires preparing arrays of cells for the handling of cells and the administration of agents. Standard multiwell microtitre plates with formats such as 6, 12, 48, 96 and 384 wells are typically used for compatibility with automated loading and robotic handling systems. Typically high throughput screens use homogeneous mixtures of agents with an indicator compound which is either converted or modified resulting in the production of a signal. The signal is measured by suitable means (for example detection of fluorescence emission, optical density, or radioactivity) followed by integration of the signals from each well containing the cells, agent and indicator compound. The present invention provides cells which have been randomly transfected or transformed with nucleic acid constructs encoding reporter molecules the expression of which is preferably under the control of said cell and is determined by the site of integration.

These clones would express reporter genes differentially in response to different stimuli. In some clones the reporter might be expressed in, for example, undifferentiated cells; in others it might only be expressed in cells differentiating along particular lineages at specific stages of differentiation, or in response to specific stimuli. Thus, a particular stimulus would be characterised by the specific subset of clones that express the reporter in response to its application. Such characterisation may include quantitative assessment of the level of reporter expression, temporal assessment of the timing of reporter expression (which could be monitored continuously in living cells following the stimulus) and also spatial assessment given that differentiation of cells may generate a heterogeneous collection of derivatives in which the reporter may be differentially expressed.

In a further preferred method of the invention said reporter molecule is a polypeptide. More preferably still said polypeptide is a polypeptide capable of fluorescence emission when excited by light. More preferably still the fluorescence emitting polypeptide is green fluorescent protein (GFP).

GFP of the jelly fish Aequorea victoria has an excitation maximum 395nm, an emission at 510nm and does not require the addition of an exogenous factor. Mutant forms of GFP are also known with altered fluorescence emission properties, see WO9821355; US5804387; US5777079; and US5625048, which are incorporated by reference.

A number of methods are known which image fluorescent cells and extract information concerning the spatial and temporal changes occuring in cells expressing fluorescent proteins, (see Taylor et al Am. Scientist 80: 322-335, 1992), which is incorporated by reference. Moreover, US5989835 and US09/031,271, both of which are incorporated by reference, disclose optical systems for determining the distribution or activity of fluorescent reporter molecules in cells for screening large numbers of agents for biological activity. The systems disclosed in the above patents also describe a computerised method for processing, storing and displaying the data generated.

In a yet further preferred method of the invention said biologically active agent is selected from the following group: polypeptides, peptides, antisense nucleic acids; ^■ double stranded RNA, peptide nucleic acids; aptamers; small biologically active compounds. In a further preferred method of the invention said polypeptide is selected from the following group of polypeptide ligands: frizzled related polypeptides (FRP); Wnt Inhibitory Factors (WIF); Dickkopf; Cerebras.

In a further preferred method of the invention said polypeptide is selected from the following group of polypeptide ligands: SFRP1; SFRP4; FRZB; SFRP2; FZD1; FZD2; FZD9; FZD3; FZD5; FZD4; FZD6; FZD7; DVL2; DVL3; GSK3B; AXTN1; AP TCFl.

hi a yet further preferred embodiment of the invention said polypeptide is selected from the following group of polypeptide ligands: human notch ligand delta-like 3 (DLL3); human notch ligand delta-like 3 precursor polypeptide; notch ligand delta- 1 (DLL1); murine notch ligand delta- like 1; human notch ligand delta-like 4 (DLL4); human notch ligand delta-like 4 (DLL4); murine notch ligand delta-like 4(DLL4); murine notch ligand delta-like 4(DLL4); murine notch ligand jagged 2; murine notch ligand jagged 2; murine notch ligand jagged 1; human notch ligand jagged 2 (JAG2); human notch ligand jagged 1 (alagille syndrome) (JAG1); murine notch ligand jagged 1; murine notch ligand delta-like 1; murine notch ligand delta-like 1.

In a further preferred method of the invention said polypeptide is a growth factor.

In a preferred method of the invention said growth factor is selected from the group consisting of: growth hormone; leptin; erythropoietm; prolactin; TNF, interleukins (IL); IL-2; IL-3; IL-4; IL-5; IL-6; IL-7; IL-9; IL-10; IL-11; IL-12; IL-13; IL-15; granulocyte colony stimulating factor; granulocyte macrophage colony stimulating factor; ciliary neuro trophic factor ; cardiotrophin-1; leukemia inhibitory factor; oncostatin M; interferon; interferons (e.g.interferon α; interferon γ); fibroblast growth factor family, the epidermal growth factor family; the bone morphogenic and T- Cell growth factor β super families; sonic hedgehog; indian hedgehog; desert hedgehog; stem cell factor (SCF). h a preferred method of the invention said agent is an antisense nucleic acid, preferably and antisense oligonucleotide.

As used herein, the term antisense oligonucleotide or antisense describes an oligonucleotide that is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide, or modified oligodeoxyribonucleotide which hybridizes under physiological conditions to DNA comprising a particular gene or to an mRNA transcript of that gene. The antisense molecules are designed so as to interfere with transcription or translation of a target gene upon hybridization with the target gene. Those skilled in the art will recognize that the exact length of the antisense oligonucleotide and its degree of complementarity with its target will depend upon the specific target selected, including the sequence of the target and the particular bases which comprise that sequence.

It is preferred that the antisense oligonucleotide be constracted so as to bind selectively with the target under physiological conditions, i.e., to hybridize substantially more to the target sequence than to any other sequence in the target cell under physiological conditions.

hi order to be sufficiently selective and potent for inhibition, such antisense oligonucleotides should comprise at least 7 (Wagner et al., Nature Biotechnology 14:840-844, 1996) and more preferably, at least 15 consecutive bases which are complementary to the target. Most preferably, the antisense oligonucleotides comprise a complementary sequence of 20-30 bases.

Although oligonucleotides may be chosen which are antisense to any region of the gene or mRNA transcripts, in preferred embodiments the antisense oligonucleotides correspond to 5' upstream sites such as translation initiation, transcription initiation or promoter sites. In addition, 3 '-untranslated regions may be targeted. The 3'- untranslated regions are known to contain cis acting sequences which act as binding sites for proteins involved in stabilising mRNA molecules. These cis acting sites often form hair-loop structures which function to bind said stabilising proteins. A well known example of this form of stability regulation is shown by histone mRNA's, the abundance of which is controlled, at least partially, post- transcriptionally.

The term antisense oligonucleotide is to be construed as materials manufactured either in vitro using conventional oligonucleotide synthesizing methods which are well known in the art or oligonucleotides synthesised recombinantly using expression vector constructs.

The present invention, thus, contemplates agents containing natural and/or modified antisense molecules that are complementary to and hybridizable with, under physiological conditions, nucleic acids encoding proteins the regulation of which results in antagonistic or agonistic effects as hereindescribed.

In a further preferred method of the invention said agent is a double stranded RNA.

A technique to specifically ablate gene function is through the introduction of double stranded RNA, (also referred to as inhibitory RNA or RNAi), into a cell which results in the destraction of mRNA complementary to the sequence included in the RNAi molecule. The RNAi molecule comprises two complementary strands of RNA (a sense strand and an antisense strand) annealed to each other to form a double stranded RNA molecule. The RNAi molecule is typically derived from exonic or coding sequence of the gene which is to be ablated.

Recent studies suggest that RNAi molecules ranging from 100-lOOObp derived from coding sequence are effective inhibitors of gene expression. Surprisingly, only a few molecules of RNAi are required to block gene expression which implies the mechanism is catalytic. The site of action appears to be nuclear as little if any RNAi is detectable in the cytoplasm of cells indicating that RNAi exerts its effect during mRNA synthesis or processing. An alternative means to generate RNAi molecules is to construct An expression cassette which includes a nucleic acid moleule comprising two parts, a first part which is derived from a gene the regulation of which is desired and a second part which is complementary to the sequence of the first part. The cassette is typically under the control of a promoter which transcribes the DNA into RNA. The complementary nature of the first and second parts of the RNA molecule results in base pairing over at least part of the length of the RNA molecule to form a double stranded hairpin RNA structure or stem-loop.

The sequences of RNAi molecules to genes which encode proteins which mediate embryonic stem cell differentiation are disclosed in currently unpublished GB0118223.7; GB0203387.6; GB0203359.5; GB0118201.3; and in published PCT application WO02/16620, the nucleic acid sequences of which are all specifically incorporated by reference.

^■ Typically, the length of the RNAi molecule is between lOObp-lOOObp. More preferably the length of RNAi is selected from lOObp; 200bp; 300bp; 400bp; 500bp;

600bρ; 700bp; 800bρ; 900bp; or lOOObp. More preferably still said RNAi is at least lOOObp. Alternatively, the RNAi molecule is between 15bp and 25bp, preferably said molecule is 21bp.

In a further preferred method of the invention said agent is an aptamer.

Conventional biological agents are small molecules, for example, peptides, polypeptides, or antibodies, which bind target molecules to produce an antagonistic or agonistic effect. It has become apparent that nucleic acid molecules also have potential with respect to providing agents with the requisite binding properties which may have utility. These nucleic acid molecules are typically referred to as aptamers. Aptamers are small, usually stabilised, nucleic acid molecules which comprise a binding domain for a target molecule. hi a yet further preferred method according to the invention said small biologically active compounds are selected from the following group: retinoic acid; hexamethylene bisacetamide; bromodeoxyuridine; lithium.

According to a further aspect of the invention there is provided an agent identified by the method according to the invention. Preferably said agent is an antagonist. Alternatively said agent is an agonist.

It will be apparent that the method according to the invention not only provides means to identify novel biologically active agents but also to identify genes involved in various biological processes such as the involvement in signal transduction pathways or in cell differentiation.

According to a yet further aspect of the invention there is provided a cell array wherein the array comprises at least one cell stably transfected/transformed with a nucleic acid molecule encoding a reporter molecule.

According to a further aspect of the invention there is provided a cell obtainable by the screening method according to the invention.

In a preferred embodiment of the invention said cell is a eukaryotic cell. Ideally said cell is an embryonic stem cell.

According to a further aspect of the invention there is provided a screening method for the isolation of a gene(s) comprising: i) providing a population of cells which have been stably transfected/transformed with a nucleic acid molecule encoding a reporter molecule; ii) cloning the transfected cells into a cell array; iii) exposing the array to at least one agent; iv) detecting a signal generated by the reporter molecule as a result of exposure to said agent; v) extracting nucleic acid from at least one cell sample comprising the cell array; and vi) determining the sequence of at least part of the genomic region into which the nucleic acid encoding the reporter molecule has integrated.

In a preferred method of the invention said method provides the further steps of (i) collating the signal (s) generated by the reporter molecule; (ii) converting the collated signal(s) into a data analysable form; and optionally (iii) providing an output for the analysed data.

According to a further aspect of the invention there is provided a method for the comparison of the biological activity of a reference agent with at least one second

/ agent comprising contacting a cell array according to the invention with an reference agent and a second, duplicate array with an agent to be tested and comparing the signal generated by the reference agent with that of the agent to be tested.

hi a preferred method of the invention said comparison comprises the steps of: i) providing a population of cells which have been stably transfected/transformed with a nucleic acid molecule encoding a reporter molecule; ii) cloning the transfected cells into a cell array; iii) preparing a duplicate array; iv) exposing an array to at least one agent to be tested; v) exposing said duplicate array to a reference agent; and vi) detecting a signal generated by the reporter molecule as a result of exposure to said agent and said reference agent.

In a further preferred method of the invention said comparison comprises the step of: (i) collating the signal(s) generated by the reporter molecule; (ii) converting the collated signal(s) into a data analysable form; and optionally (iii) providing an output for the analysed data.

According to a further aspect of the invention there is provided a vector comprising: a reporter molecule; a splice acceptor site and an internal ribosome entry site characterised in that said splice acceptor and said internal ribsome entry site are operably linked to facilitate expression of said reporter molecule.

Preferably said splice acceptor is positioned 5' to an internal ribosome entry site.

It will be apparent that a vector can randomly integrate into a genome. The provision of a splice acceptor operably linked to an internal ribosome entry site will reduce the likelihood of a reporter molecule integrating out of frame thereby increasing the efficiency of the screening method.

In a preferred embodiment of the invention said vector further includes a nucleic acid which encodes a selectable marker.

In a further preferred embodiment of the invention vector includes a nucleic acid molecule which encodes a green fluorescent protein reporter molecule.

It will be apparent to one skilled in the art that the gene screening method may use biologically active agents identified by the screening method according to the invention; known biologically active agents (e.g. retinoic acid; hexamethylene bisacetamide; bromodeoxyuridine; lithium); nucleic acid molecules as disclosed in GB0118223.7; GB0203387.6; GB0203359.5; GB0118201.3; and in published PCT application WO02/16620 and polypeptides as disclosed in PCT/GB02/01195. It will also be apparent to the skilled artsan that the comparison method allows the identification of agents which have common gene targets and/or signal transduction pathways. An embodiment of the invention will now be described by example only and with reference to the following figures, materials and methods.

Figure 1 is the nucleic acid sequence of murine notch ligand delta-like 1;

Figure 2 is the amino acid sequence of murine notch ligand delta-like 1;

Figure 3 is the nucleic acid sequence of murine notch ligand jagged 1;

Figure 4 is the nucleic acid sequence. of human notch ligand jagged 1 (alagille syndrome) (JAG1);

Figure 5 is the amino acid sequence of human notch ligand jagged 1 (alagille syndrome);

Figure 6 is the nucleic acid sequence of human notch ligand jagged 2 (JAG2)

Figure 7 is the amino acid sequence of human notch ligand jagged 2 (JAG2);

Figure 8 is the amino acid sequence of murine notch ligand jagged 1;

Figure 9 is the nucleic acid sequence of murine notch ligand jagged 2;

Figure 10 is the amino acid sequence of murine notch ligand jagged 2;

Figure 11 is the nucleic acid sequence of human notch ligand delta-like 3 (DLL3);

Figure 12 is the amino acid sequence of human notch ligand delta-like 3 precursor polypeptide;

Figure 13 is the nucleic acid sequence of human notch ligand delta-1 (DLL1); Figure 14 is the amino acid sequence of murine notch ligand delta- like 1;

Figure 15 is the nucleic acid sequence of human notch ligand delta-like 4 (DLL4);

Figure 16 is the amino acid sequence of human notch ligand delta-like 4 (DLL4);

^' Figure 17 is the nucleic acid sequence of murine notch ligand delta-like 4(DLL4);

Figure 18 is the amino acid sequence of murine notch ligand delta-like 4(DLL4);

Figure 19 represents the nucleic acid sequence of human Wnt 13;

Figure 20 represents the nucleic acid sequence of human dickkopfl;

Figure 21 represents the nucleic acid sequence of human dickkopfl;

Figure 22 represents the nucleic acid sequence of human dickkopfl; and

Figure 23 represents the nucleic acid sequence of human dickkopf4;

Figure 24 represents the nucleic acid sequence of WNT-1;

Figure 25 represents the amino acid sequence of WNT-1;

Figure 26 represents the nucleic acid sequence of WNT-2;

Figure 27 represents the amino acid sequence of WNT-2;

Figure 28 represents the nucleic acid sequence of WNT 2B; Figure 29 represents the amino acid sequence of WNT 2B;

Figure 30 represents the nucleic acid sequence of WNT 3;

Figure 31 represents the amino acid sequence of WNT 3;

Figure 32 represents the nucleic acid sequence of WNT 4;

Figure 33 represents the amino acid sequence of WNT 4;

Figure 34 represents the nucleic acid sequence of WNT 5A;

Figure 35 represents the amino acid sequence of WNT 5 A;

Figure 36 represents the nucleic acid sequence of WNT 6;

Figure 37 represents the amino acid sequence of WNT 6;

Figure 38 represents the nucleic acid sequence of WNT 7A;

Figure 39 represents the amino acid sequence of WNT 7 A;

Figure 40 represents the amino acid sequence of WNT 7B;

Figure 41 represents the nucleic acid sequence of WNT 8B;

Figure 42 represents the amino. acid sequence of WNT 8B;

Figure 43 represents the nucleic acid sequence of WNT 10B;

Figure 44 represents the amino acid sequence of WNT 10B; Figure 45 represents the nucleic acid sequence of WNT 11;

Figure 46 represents the amino acid sequence of WNT 11;

Figure 47 represents the nucleic acid sequence of WNT 14

Figure 48 represents the amino acid sequence of WNT 14;

Figure 49 represents the nucleic acid sequence of WNT 16;

Figure 50 represents the amino acid sequence of WNT 16;

Figure 51 represents the nucleic acid sequence of FZD 1;

Figure 52 represents the amino acid sequence of FZD 1;

Figure 53 represents the nucleic acid sequence of FZD 2;

Figure 54 represents the amino acid sequence of FZD 2;

Figure 55 represents the nucleic acid sequence of FZE 3;

Figure 56 represents the amino acid sequence of FZE 3;

Figure 57 represents the nucleic acid sequence of FZD 4;

Figure 58 represents the amino acid sequence of FZD 4;

Figure 59 represents the nucleic acid sequence of FZD 5; Figure 60 represents the amino acid sequence of FZD 5;

Figure 61 represents the nucleic acid sequence of FZD 6;

Figure 62 represents the amino acid sequence of FZD 6;

Figure 63 represents the nucleic acid sequence of FZD 7;

Figure 64 represents the amino acid sequence of FZD 7;

Figure 65 represents the nucleic acid sequence of FZD 8;

Figure 66 represents the amino acid sequence of FZD 8;

Figure 67 represents the nucleic acid sequence of FZD 9;

Figure 68 represents the amino acid sequence of FZD 9;

Figure 69 represents the nucleic acid sequence of FZD 10;

Figure 70 represents the amino acid sequence of FZD 10;

Figure 71 represents the nucleic acid sequence of FRP;

Figure 72 represents the amino acid sequence of FRP;

Figure 73 represents the nucleic acid sequence of S ARP 1 ;

Figure 74 represents the amino acid sequence of S ARP 1;

Figure 75 represents the nucleic acid sequence of SARP 2; Figure 76 represents the amino acid sequence of SARP 2;

Figure 77 represents the nucleic acid sequence of FRZB;

Figure 78 represents the amino acid sequence of FRZB;

Figure 79 represents the nucleic acid sequence of FRPHE;

Figure 80 represents the amino acid sequence of FRPHE;

Figure 81 represents the nucleic acid sequence of SARP 3;

Figure 82 represents the amino acid sequence of SARP 3;

Figure 83 represents the nucleic acid sequence of CER 1;

Figure 84 represents the amino acid sequence of CER 1 ;

Figure 85 represents the nucleic acid sequence of DKTCl ;

Figure 86 represents the amino acid sequence of DKTCl;

Figure 87 represents the nucleic acid sequence of DKK 2;

Figure 88 represents the amino acid sequence of DKK 2;

Figure 89 represents the nucleic acid sequence of DKK 3;

Figure 90 represents the amino acid sequence of DKK 3; Figure 91 represents the nucleic acid sequence of DKK 4;

Figure 92 represents the amino acid sequence of DKK 4;

Figure 93 represents the nucleic acid sequence of WTF-1;

Figure 94 represents the amino acid sequence of WIF-1;

Figure 95 represents the nucleic acid sequence of SRFP 1;

Figure 96 represents the amino acid sequence of SRFP 1;

Figure 97 represents the nucleic acid sequence of SRFP 4;

Figure 98 represents the amino acid sequence of SRFP 4; and

Figure 99 illustrates a vector construct for use in the method according to the invention.

Materials and Methods

Transfection Vectors

Enhancer, gene and promoter trap vectors all have the following basic properties. A bacterial selection marker (eg ampicilin resistance) with appropriate control sequences to allow replication of the vector for cloning purposes and expression of selectable markers. In addition all three types of vector will have mammalian selection cassette eg neomycin resistance and a reporter cassette eg GFP. In these vectors the mammalian selection cassette may include an antibiotic resistance gene, such as the neomycin resistance gene under the control of a separate, constitutive promoter (e.g the Major Immediate Early Promoter of human Cytomegalovirus), or it might be linked to the reporter cassette by an internal ribosome entry site (IRES) such that it would be under the control of the endogenous gene into which it is inserted in the transfected mammalian cell, or it might be linked to the reporter gene in a way that results in translation of a fusion protein between the reporter and the selection gene.

i) Enhancer Trap (see, Gossler A., Joyner A.L., Rossant J., SkamesW.C. (1989). Science 244, 463; and Korn R., Schoor M., Neuhaus H., Henseling U., Soininen R., Zachgo J., Gossler A. (1992). Mech. Dev. 39, 95).

A minimal promoter for your cell of interest (in EC/ES Thymidine kinase promoter or mouse hsp60) driving a reporter cassette (GFP) with a polyadenylation signal.

ii) Gene Trap (see Friedrich G., Soriano P. (1991). Genes Dev. 5, 1513)

Splice acceptor site with a branch site 30 bps upstream linked to a reporter with a polyadenylation signal. Modifications can include splice acceptor sites in all three reading frames for the reporter cassette.

iii) Promoter Trap (see, Reddy S., DeGregory J.V., von Melchner H., Ruley

H.E. (1991). J. Virol. 65, 1507)

The same as gene trap but no splice acceptor site.

Figure 99 illustrates an alternative trap vector. A splice accepting (SA) site (GTCCCAGGTCCCGAAAA) was attached to the 5' end of an Internal Ribosome Entry Site (IRES) sequence by PCR cloning. The S A-IRES sequence was then cloned into the BamHI site of pd2EGFP-l (Clontech). pd2EGFP-l contains a NEO selection marker driven a separate SV40 promoter, permitting selection of transfected clones. Illustration of The Three Methods of Vector Design for detection of Gene activation Events.

Enhancer Trap

ATG

Selection

Minimal Promoter eg Neomycin Promoter

resistance

Gene

Branc

Site (30 bps 5' to Splice acceptor)

Promoter Trap

PolyA Selection

GFP Promoter signal e.g Neomycin resistance

Maintenance of cell lines

All cells were grown in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% by volume foetal calf serum (Gibco BRL) and 2mM L- glutamine. Tissue culture flasks were incubated in a humidified atmosphere of 10% CO₂ in air at 37°C.

Transfection of human ES cells

Human ES cells were cultured in "Knock-Out" DMEM (GICO Life Technologies Ltd) supplemented with 20% Serum Replacement (GICO Life Technologies Ltd) at 37°C under a humidified atmosphere of 5% CO₂ in air, on mitomycin C inactivated mouse embryo fibroblasts (Thomson et al 1998). The cells were harvested by exposure for 3 min at 37 C to a solution of 0.05% trypsin in calcium and magnesium free Dulbecco's Phosphate Buffered saline containing 1 mM EDTA. When the cells detached they were resuspended in fresh medium and plated into a fresh tissue culture vessel, precoated with a l:30dilution of Matrigel (Becton Dickinson), at 6 x 10⁴ cells per cm². After incubation at 37 C overnight the cells were transfected, following the manufacturer's protocol, as follows: 9.5 μg DNA was diluted into 300 μl 0.15 M NaCI and mixed with 21 μl ExGen 500 (MBI Fermentas) and vortexed. After incubating at room temperature for 10 min, the DNA: ExGen solution was mixed with 3 ml culture medium and added a culture of ES cells as described above in a 35 mm (9.6 cm²) dish. After incubating for 6 - 12 hours at 37 C, the medium was removed and medium containing an appropriate selection agent (e.g. G418) was added to the cells. Cultures were fed every 2 - 3 days, and colonies isolated and expanded after 2 - 3 weeks.

Treatment of NTERA2 Cells Retinoic acid

NTERA2 EC cells were induced to differentiate with retinoic acid as previously described (Andrews 1984). Medium was aspirated from confluent flasks of EC cells and the cells rinsed in sterile PBS. 1ml of 0.25% (w/v) trypsin in 2mM EDTA was added per 75cm² flask and the flask incubated at room temperature for .up to 5 minutes. Vigorous shaking was subsequently used to dislodge the cells. Cells "were suspended in 9ml of supplemented DMEM per ml of trypsin used and counted in a haemocytometer. Cells were seeded at 10⁶ cells per 75 cm² flask, in medium containing 10^" M all-trα7W-retinoιc acid (Eastman Kodak), diluted from a 10^" M stock solution in dimethyl sulfoxide (DMSO). Flasks were incubated as described above and the media replaced as and when required.

Hexamethylene bisacetamide (HMBA) Cells to be treated with HMBA were prepared as described for retinoic acid, but grown in medium supplemented with 10^"3M HMBA instead of RA, as described by Andrews et al (1990).

Harvesting of cells Cells were dislodged from the culture vessel with trypsin and suspended in 9ml culture medium per ml of trypsin solution used, as described above. The cell suspension was then centrifuged at 400 x g for 3 minutes and the medium aspirated from the resulting cell pellet. Cells were then rinsed in 5ml PBS and centrifuged again at 400 x g for 1 minute. The PBS rinse was aspirated and the cells stored at - 80°C or used immediately.

Cloning Strategy for Creation of Cell Arrays

After transfection with the GFP gene trap vector, the cells were cultured in the presence of 400 ug /ml G418. Only cells that have incorporated the selectable marker neoR, formed colonies. After 2-3 weeks, individual colonies were picked manually using a micropipet, and grown up individually. Whether the GFP insert was expressed was assessed for each colony by examination using a UV microscope, before and after differentiation induced with retinoic acid. A series of colonies were then randomly selected and individually seeded out, one per well of a set of 24 well plates. Replicate arrays of sets of colonies were produced by seeding into multiple plates. Each replicate was then exposed to medium containing a diffeernt inducing chemical agent, e.g. all-trans retinoic acid, 13-cid retinoic acid, 9-cis retinoic acid and HMBA. At successive time points the fluorescence from each colony in the array was asssessed.

Strategies for Forming Cell Arrays

Example 1

Cell arrays of transfected cells would be maintained in separate cultures of the different clones of cells, each clone having the reporter gene inserted into a different site or sites in the genome.

During screening, cell arrays would be set up in multiwell plates by transferring from the stock cultures a different clone of cells to each well of the set of plates constituting the array. Multiple replicates of the array could be established in different sets of plates. The multiwell plates might be of the standard format comprised of 6, 12, 24, 48, 96, or 384 wells, or in the format of 'Terasaki' microwell plates.

Example 2

An alternative would be to spot individual cells, or small numbers of cells, from each clone onto the surface of a single 'vessel', so that a set of separate clones growing on a surface are bathed in a common medium. The 'vessel' might be any cell culture vessel with a suitable surface for the culture of cells - for example a standard 35 mm, 60 mm or 100 mm diameter tissue culture dish. Alternatively, individual colonies maybe established in the wells of a multi- well plate as described in example 1. Cells of interest could then be physically removed from a colony. Cells showing interesting expression patterns could then be isolated and expanded in a separate culture container. Example 3

A further alternative is to maintain the library of transfected cells as a common pool rather than as a set of isolated clones. To set up cell arrays, single cells from the libraries would be spotted into wells (as in example 1 above) or onto arrays on a surface of a single vessel (as in 2 above). Once the cells had grown up to form individual colonies these would be replica plated. In the case of growth in wells, cells would be harvested from wells using standard tissue culture protocols and re- seeded to multiple wells to allow formation of replicate arrays. In the case of cell arrays on a single surface, colonies might be replicated in a variety of ways. For example, carefully placing an adherent membrane of the array of colonies will enable small number of cells from each colony to transfer to a new culture surface to form a replica array. Alternatively, each colony would be dispersed individually in a micropipette and transferred to a series of separate spots forming multiple replicate arrays; such a procedure could be automated by robotic techniques as herein disclosed.

Claims

1 A screening method for the identification of biologically active agents comprising: i) providing a population of cells which have been stably transfected with a nucleic acid molecule encoding a reporter molecule; ii) cloning the transfected cells into a cell array; iii) exposing the array to at least one agent to be tested; and iv) detecting a signal generated by the reporter molecule as a result of exposure to said agent.

2. A method according to Claim 1 which has the further steps of: i) collating the signal (s) generated by the reporter molecule; ii) converting the collated signal(s) into a data analysable form; and optionally iii) providing an output for the analysed data.

3. A method according to Claim 1 or 2 wherein said cells are eukaryotic.

4. A method according to Claim 3 wherein said eukaryotic cells are: protozoan; fungal; or mammalian.

5. A method according to Claim 4 wherein said protozoan cell is selected from the group consisting of: Plasmodium spp; Plasmodium falciparum; Leishmania spp; Leishmania major; Trypansoma brucei spp; Trypanosoma brucei brucei; Trypanosoma brucei rhodesiensis; Trypanosoma cruzi; Giardia spp.; Cryptosporidium spp. Acanthamoeba spp.; Babesia spp.; Babesia bovis; Toxoplasma spp; Entamoeba spp. Naegleria spp.

A method according to Claim 4 wherein said cell is a fungal cell.

6. A method according to Claim 5 wherein said fungal cell is selected from the group consisting of: Saccharomyces cerevisiae; Candida spp.; Candida albicans.

1. A method according to Claim 1 or 2 wherein said cell is prokaryotic.

8. A method according to Claim 7 wherein said cell is selected from the group consisting of: Staphylococcus aureus; Staphylococcus epidermidis; Enterococcus faecalis; Mycobacterium tuberculsis; Mycobacterium bovis; Mycobacterium leprae;

Streptococcus group B; Streptoccocus pneumoniae; Streptoccocus equi; Streptoccocus pyogenes; Streptoccocus coelicolor; Helicobacter pylori; Neisseria gonorrhea; Streptococcus group A; Borrelia burgdorferi; Coccidiodes immitis;

Histoplasma sapsulatum; Neisseria meningitidis type B; Neisseria meningitidis type

C Shigella flexneri; Escherichia coli; Haemophilus influenzae; Bacteriodes fragilis;

Bordetellabronchiseptica; Bordetella parapertussis; Bordetella pertussis; Burkholderia psuedomallei; Campylobacter je'juni; Clostridium difficile;

Cornybacterium diphtheria; Salmonella typhi; Yersinia enterocolitica; Yersinia pestis.

9. A method according to Claim 4 wherein said cell is mammalian.

10. A method according to Claim 9 wherein said cell is derived from primary cell-line.

11. A method according to Claim 10 wherein said primary cell line is selected from the group consisting of: fibroblasts, keratinocytes, endothehal cells, renal tubule cells, neural cells; hepatocytes; or stem cells.

12. A method according to Claim 11 wherein said cells are stem cells.

13. A method according to Claim 12 wherein said stem cell is selected from the group consisting of: haemopoietic stem cell; neural stem cell; bone stem cell; muscle stem cell; embryonic stem cell; embryonic germ cell; mesenchymal stem cell; trophoblastic stem cell; epithelial stem cell (derived from organs such as the skin, gastrointestinal mucosa, kidney, bladder, mammary glands, uterus, prostate and endocrine glands such as the pituitary); endodermal stem cell (derived from organs such as the liver, pancreas, lung and blood vessels); embryonal carcinoma cell.

14. A method according to Claim 13 wherein said stem cell is a human embryonic stem cell.

15. A method according to any of Claims 9-14 wherein said agent is a polypeptide and is selected from the group consisting of: frizzled related polypeptides (FRP); Wnt Inhibitory Factors (WJJF); Dickkopf; Cerebras.

16. A method according to Claim 15 wherein said polypeptide is selected from the group represented by the amino acid sequences comprising: Fig 2; Fig 5; Fig 7 Fig 8; Fig 10; Fig 12; Fig 14; Fig 16; Fig 18; Fig 25; Fig 27; Fig 29; Fig 31; Fig 33 Fig 35; Fig 37; Fig 39; Fig 40; Fig 42; Fig 44; Fig 46; Fig 48; Fig 50; Fig 52; Fig 54 Fig 56; Fig 58; Fig 60; Fig 62; Fig 64; Fig 66; Fig 68; Fig 70; Fig 72; Fig 74; Fig 76 Fig 78; Fig 80; Fig 82; Fig 84; Fig 86; Fig 88; Fig 90; Fig 92; Fig 94; Fig 96; Fig 98 or active binding fragment thereof.

18. A method according to any of Claims 1 -14 wherein said agent is a double stranded RNA.

19. A method according to Claim 18 wherein said double stranded RNA is derived from the the nucleic acid molecules represented by the sequences in the group comprising: Fig 1; Fig 3; Fig 4; Fig 6; Fig 9; Fig 11; Fig 13; Fig 15; Fig 17; Fig 19; Fig 20; Fig 21; Fig 22; Fig 23; Fig 24; Fig 26; Fig 28; Fig 30; Fig 32; Fig 34; Fig 36; Fig 38; Fig 41; Fig 43; Fig 45; Fig 47; Fig 49; Fig 51; Fig 53; Fig 55; Fig 57;

Fig 59; Fig 61; Fig 63; Fig 65; Fig 67; Fig 69; Fig 71; Fig 73; Fig 75; Fig 77; Fig 79;

Fig 81; Fig 83; Fig 85; Fig 87; Fig 89; Fig 91; Fig 93; Fig 95; Fig 97; or fragment thereof.

20. A method according to any of Claims 9-14 wherein said agent is a polypeptide and is selected from the group consisting of: growth hormone; leptin; erythropoietm; prolactin; tumour necrosis factor; interleukins (IL); IL-2; IL-3; IL-4;

IL-5; IL-6; IL-7; IL-9; IL-10; IL-11; IL-12; IL-13; IL-15; granulocyte colony stimulating factor; granulocyte macrophage colony stimulating factor; ciliary neuro trophic factor ; cardiotrophin-1; leukemia inhibitory factor; oncostatin M; interferon; interferons (e.g.interferon α; interferon γ); fibroblast growth factor family, the epidermal growth factor family; the bone morphogenic and T- Cell growth factor β super families; sonic hedgehog; indian hedgehog; desert hedgehog; stem cell factor.

21. A method according to any of Claims 1-14 or 19 wherein said agent is an antisense nucleic acid.

22. A method according to any of Claims 1-14 wherein said agent is an aptamer.

23. A method according to any of Claims 9-14 wherein said agent is selected from the group consisting of: retinoic acid; hexamethylene bisacetamide; bromodeoxyuridine; lithium.

24. An agent identified by the method according to any of Claims 1-14.

25. A cell obtainable by the method according to any of Claims 1-22.

26. A cell according to Claim 25 wherein said cell is an embryonic stem cell.

27. A cell array obtainable by the method according to any of Claims 1 -22.

28. A screening method for the isolation of a gene comprising: i) providing a population of embryonic stem cells which have been stably transfected/transformed with a nucleic acid molecule encoding a reporter molecule; ii) cloning the transfected cells into a cell array; iii) exposing the array to at least one agent; iv) detecting a signal generated by the reporter molecule as a result of exposure to said agent; v) extracting nucleic acid from a cell sample comprising the cell array; and vi) determining the sequence of at least part of the genomic region into which the nucleic acid encoding the reporter molecule has integrated.

29. A method for the comparison of the biological activity of a reference agent with at least one other agent comprising the steps of: i) providing a population of cells which have been stably transfected/transformed with a nucleic acid molecule encoding a reporter molecule; ii) cloning the transfected cells into a cell array; iii) preparing a duplicate array; iv) exposing said cell array to at least one agent to be tested; v) exposing said duplicate array to a reference agent; and vi) detecting a signal generated by the reporter molecule as a result of exposure to said agent and said reference agent.

30. A method according to Claim 29 wherein of the invention said comparison comprises the step of:

(i) collating the signal(s) generated by the reporter molecule; (ii) converting the collated signal(s) into a data analysable form; and optionally (iii) providing an output for the analysed data.

31. A vector comprising: a reporter molecule; a splice acceptor site and an internal ribosome entry site characterised in that said splice acceptor and said internal ribsome entry site are operably linked to facilitate expression of said reporter molecule.

32. A vector according to Claim 31 wherein said splice acceptor is positioned 5' to an internal ribosome entry site.

33. A vector according to Claim 31 or 32 wherein said vector includes a nucleic acid molecule which encodes a selectable marker.

34. A vector according to any of Claims 31-33 wherein said vector includes a nucleic acid molecule which encodes a green fluorescent protein reporter molecule.

35. Use of a vector according to any of Claims 31-34 in the methods according to Claim 1-23 or 28-30.