WO2009029055A1

WO2009029055A1 - P53 isoform gene transgenic non-human animal

Info

Publication number: WO2009029055A1
Application number: PCT/SG2008/000322
Authority: WO
Inventors: Jinrong Peng; Jun Chen; David P. Lane
Original assignee: Agency For Science, Technology And Research
Priority date: 2007-08-31
Filing date: 2008-09-01
Publication date: 2009-03-05

Abstract

There is provided a transgenic non-human animal, wherein the transgenic non- human animal genome comprises at least one stably integrated reporter gene operably linked to regulatory elements of a p53 isoform gene, wherein the p53 isoform gene is capable of modulating the activity of p53. In particular, the transgenic non-human animal is a transgenic fish and the p53 isoform gene is Δ113p53 gene and the regulatory elements are the Δ113p53 gene's regulatory elements. There are also provided uses of the transgenic non-human animal according to the invention.

Description

p53 ISOFORM GENE TRANSGENIC NON-HUMAN ANIMAL

Field of the invention

The present invention relates to transgenic non-human animal(s). In particular, the present invention relates to a transgenic fish. More in particular, there is provided a transgenic non-human animal for use in detecting and/or monitoring nucleic acid damages and/or carcinogenic agent in the environment. Further the transgenic fish may be used in screening for effective drugs for cancer treatment.

Background of the invention

Non-human animal models of disease states play an important role in identifying the underlying biochemical mechanisms of particular diseases, like cancer, as well as discovering therapeutic agents to eradicate the disease or otherwise lessen its symptoms. Working with large animals however poses several drawbacks. Many of the animals used in such models are relatively large vertebrates which take up a large amount of research space, are costly to feed and otherwise maintain, have slow reproductive cycles, produce relatively few offspring at one time, and cannot effectively mimic all desired disease states.

Transgenic technology involves the transfer of a foreign gene into a host organism enabling the host to acquire a new and inheritable trait. The technique involves injecting foreign DNA into fertilized eggs and many animals that develop from the injected eggs retain the foreign DNA. This technology has been used to create transgenic mice, sheep, pigs etc. The transgenic approach provides to animals new and beneficial traits. For example, the transgenic animals can be used as bioreactors to produce commercially useful compounds by expression of a useful foreign gene in milk or in blood. Many pharmaceutically useful protein factors have been expressed in this way. Transgenic mice have been widely used in medical research, particularly in the generation of transgenic animal models for human disease studies.

Fish are also an intensive research subject of transgenic studies. There are many ways of introducing a foreign gene into fish, including: microinjection, electroporation, sperm-mediated gene transfer, gene bombardment or gene gun, liposome-mediated gene transfer, and the direct injection of DNA into muscle tissue. The zebrafish, Danio rerio, is a new model organism for vertebrate developmental biology. As an experimental model, the zebrafish offers several major advantages such as easy availability of eggs and embryos, tissue clarity throughout embryogenesis, external development, short generation time and easy maintenance of both the adult and the young. Therefore an appropriate animal model will be of interest for understanding cellular and molecular genetic features of various disease states such as the cancers. An appropriate animal model would be invaluable to elucidate the multistep process of genetic mutations, to develop more effective drugs, as well as a model to monitor the presence of carcinogens in the aquatic environment.

In more than 50% of the human tumors, the p53 gene is mutated, and the loss of p53 mediated apoptosis and cell cycle control has been implicated as an important event in tumor progression. Activation of p53, either at the transcriptional level or protein modification level, induces expression of many genes that form a network to regulate cell cycle or cell apoptosis. This network initiated from p53 is essential to eliminate abnormal cells (especially cancer- predisposing cells) and maintain normal cells. Thus, p53 is a crucial factor in suppressing tumorogenesis. Enhancing p53 activity in tumour cells is thought to be an efficient way to kill cancer cells. However, practically, in many cases enhancing p53 activity is not correlated to cancer cell death and the reason is unknown. Besides, many patients cannot tolerate or are physically not suitable to take radiotherapy or chemotherapy, two commonest ways used in cancer treatment. Therefore, tremendous efforts have been spent to screen for more effective drugs that can be used in cancer treatment.

A recent development in p53 study is the discovery of p53 isoforms initiated from the alternative promoter in intron 4 (e.g Δ133p53 in human and Δ113p53 in zebrafish) (Bourdon et al., 2005; Chen et al., 2005). The Δ133p53/Δ113p53 proteins are N-terminal truncated forms of p53 with deletion of both the Mdm2- interacting motif and transcription activation domain together with partial deletion of the DNA-binding domain. However, the dimerization domain is intact in Δ133p53/Δ113p53. Preliminary analyses have suggested that DNA fragment containing intron 4 of human p53 could drive the expression of the reporter gene luciferase in H1299 cells. The authors also suggested that Δ133p53 might act as a dominant negative regulator of p53 since co-transfection p53 with Δ133p53 impaired p53-induced cell apoptosis (Bourdon et al., 2005). However, many questions as to how the A133p53 /A113p53 expression regulated and what are their biological functions remain unanswered. Further, it would be of interest to understand the functional relationship between p53 and Δi33p53/ΔH3p53.

Summary of the invention

The present invention addresses the problems above, and in particular to provide a new transgenic animal, wherein the transgenic non-human animal genome comprises at least one stably integrated reporter gene operably linked to regulatory elements of the p53 isoform gene, wherein the p53 isoform gene is capable of modulating the activity of p53. In particular, the reporter gene may be operably linked to the 5'-upstream region of a p53 isoform gene. The 5'- upstream region of the p53 isoform gene may comprise the p53 isoform gene regulatory elements. However, the reporter gene may also be operably linked to other regulatory elements, for example enhancers, placed outside the 5'- upstream region of the p53 isoform gene. In particular, the transgenic non- human animal is a transgenic fish, more in particular a transgenic zebrafish. The p53 isoform gene may be the Δ113p53 gene and the regulatory elements may be the Δ113p53 gene's regulatory elements. According to a particular aspect, the transgenic non-human animal is a transgenic fish and the p53 isoform gene is Δ113p53 gene and the regulatory elements are the Δ113p53 gene's regulatory elements. However, the present invention is not limited to transgenic fish. Accordingly, any gene homologous to the Δ113p53 gene and/or their regulatory elements, capable of modulating the expression and/or activity of p53 may be used for producing non-human transgenic animals according to the invention.

The regulatory element may comprise a 4113bp 5'-upstream region, or portion thereof, of the translation start site of the p53 isoform gene; a 5'-upstream region -1041 to -1991bp, or portion thereof, of the transcription start site of the p53 isoform gene; and/or a 5'-upstream region -1 to -239bp, or portion thereof, of the transcription start site of the p53 isoform gene according to the invention. The regulatory element may comprise a promoter.

The reporter gene may be expressed at control expression levels. An increased expression of the reporter gene, compared to the control expression levels, may be induced by exposure of the animal (in particular, a fish) to nucleic acid (including but not limited to DNA) damaging and/or carcinogenic agent(s). In particular, an increased reporter gene expression, compared to the control expression level(s), is indicative of increase in the expression of the p53 isoform gene, in at least one cell. In particular, the increased reporter gene expression, compared to the control expression level(s), is indicative of increase in the expression of the p53 isoform gene according to the invention, in at least one cell. More in particular, the increased expression of the p53 isoform gene prevents, reduces and/or inhibits the p53 expression and/or activity in at least one cell. Further, an increased expression of the p53 isoform gene prevents apoptosis and/or induces cancer progression in at least one cell.

The reporter gene may comprise a transcription stop-site. The reporter gene may be selected from the group consisting of luciferase, galactosidase, chloramphenicol, acetyltransferase, b-glucuronidase, and alkaline phosphatase. In particular, the reporter gene is a fluorescent protein gene, for example, a fluorescent protein selected from the group consisting of GFP, RFP, BFP, YFP, and dsRED2. More in particular, the fluorescent protein is GFP.

The non-human animal according to the invention may be any animal suitable for the purpose of the present invention. In particular, the non-human transgenic animal is a transgenic fish. The transgenic fish according to the invention may be any fish, in particular a transgenic zebrafish. More in particular, the p53 isoform gene is the Δ113p53 gene and the regulatory elements are the Δ113p53 gene's regulatory elements.

The present invention also provides a method of detecting and/or monitoring nucleic acid (example, DNA) damaging and/or carcinogenic agents in the environment comprising: a) contacting and/or exposing a transgenic fish to the environment, wherein the transgenic non-human animal (for example, a transgenic fish) genome comprises at least one stably integrated reporter gene operably linked to a p53 isoform gene regulatory elements, wherein the p53 isoform gene is capable of modulating the expression and/or activity of p53; b) determining the reporter gene expression, wherein the increased reporter gene expression compared to control reporter gene expression is indicative of the of presence of nucleic acid damaging and/or carcinogenic agents in the environment. There is also provided a method of screening drugs and/or agents capable of decreasing and/or inhibiting the expression of a p53 isoform gene, wherein the p53 isoform gene is capable of modulating the activity of p53, comprising a) contacting and/or exposing a transgenic fish to a test drug and/or agent, wherein the transgenic non-human animal (for example, a transgenic fish) genome has stably integrated a reporter gene operably linked to the p53 isoform gene regulatory elements; and b) determining if the test drug and/or agent is capable of decreasing and/or inhibiting the expression of the p53 isoform gene.

The step b) may comprise determining if the test drug and/or agent is capable of decreasing and/or inhibiting the expression of the reporter gene.

The test drug and/or agent may be a nucleic acid molecule capable of binding and/or hybridising to the p53 isoform gene, mRNA, promoter and/or a portion thereof. In particular, the test drug and/or agent may be a nucleic acid molecule comprising a nucleotide complementary to and/or which hybridises to the p53 isoform gene, mRNA, promoter and/or a portion thereof. The nucleic acid molecule may be an antisense nucleic acid molecule, for example, an antisense single strand RNA (sRNA), double strand RNA (dsRNA), double strand DNA (dsDNA), double strand hybrid RNA/DNA (RNA/DNA), small interfering RNA (siRNA), micro RNA (miRNA), morpholinos (MO/PMO) and/or ribozymes.

Brief description of the figures

Figure 1. Diagram showing the genomic structure of the zebrafish p53 gene and the relative position of the 4.113 kb genomic DNA fragment cloned for the Δ113p53 promoter activity analyses. The zebrafish p53 gene has 10 exons (light blue or grey box) and 9 introns (black lines linking boxes). The start codon ATG of p53 is located in the second exon. The lengths of intron 1-4 of p53 are 637 bp, 240 bp, 92 bp and 2692, respectively, as shown in the diagram. Transcription of Δ113p53 starts in the intron 4 (dark blue/grey box ES) and the mature Δ113p53 transcript contains 155 bp intron 4 sequence joined to the exon 5 of p53 after splicing an intron of length 842 bp (intron 1 for Δ113p53). The start codon ATG of Δ113p53 is located in the exon 5 of p53. The 4.113 kb genomic DNA fragment cloned is immediately upstream of the Δ113p53 start codon ATG and ends in the exon 1 of p53, thus it excludes the p53 promoter sequences. The 4.113 kb DNA fragment was cloned into the pEGFP vector to generate the Δ113p53:gfp plasmid.

Figure 2. Generation of Tg(Δ113p53:gfp) transgenic fish. (A) Plasmid DNA injection induced Δ113p53 expression in the injected WT zebrafish embryos. Uninjected and buffer-injected embryos were used as the controls. (B) Gfp fluorescence observed in the embryos injected with the Δ113p53::gfp plasmid.. (C) To generate the Tg(Δ113p53:gfp) transgenic fish, the Δ113p53:gfp plasmid was linearized and injected into single-cell stage embryos and individual fish were screened based on gfp fluorescence as described in 'Materials and methods' section. The homozygous Tg(Δ113p53:gfp) transgenic fish were mated with def"⁴²⁹ heterozygous fish and their F2 progenies were examined for Gfp fluorescence and genotyped for def"⁴²⁹ mutation. At 1 dpf and 2 dpf, Gfp expression was weakly expressed in WT (def+/+) and the heterozygotes (def+/- ) siblings (sb) whilst the def¹'⁴²⁹ homozygous embryos (def-/-) had strong and ubiquitous Gfp expression in the head region. (D) At 3 dpf, progenies exhibiting strong Gfp fluorescence in the head and digestive organs (the upper embryo) were found to be def¹¹⁴²⁹ homozygotes (def -/-) whilst the rest showing ubiquitous weak Gfp fluorescence (the lower embryo) were either WT (def+/+) or def +/- siblings. (E). Example of genotyping the F2 progenies in C and D using primers specific for def, lacz and gfp. Figure 3. Diagram showing the details of each Δ113p53 promoter deletion constructs. The Δ113p53:gfp plasmid is designated as PO and the transcription start site (TSS) of Δ113p53 as the position +1. The start position of intron 4 of p53 is also highlighted (-1692). The nucleotide position +1054 is immediately 5'- upstream of the start codon ATG of Δ113p53. Total nine deletion plasmids (P1 to P9) were constructed, including five plasmids with 5'-deletions (P1 to P5), two with 3'-deletions (P7 and P8) and two with internal deletions (P6 and P9).

Figure 4. Promoter activity test for each Δ113p53:gfp deletion plasmid. (A) PO- P9 plasmid DNAs were injected into one-cell stage WT embryos and Gfp fluorescence in each test was visualized under a fluorescence microscope at 24 hours post-injection. (B) The levels of gfp transcripts in each test were examined via RNA gel blot hybridization using a gfp specific probe (top panel).

The 18S rRNA was used as the loading control (second panel). The levels of the endogenous Δ113p53 expression in the same samples were examined via semi-quantitative RT-PCR. The elongation factor a gene (elfa) was used as the control for RT-PCR. WT: uninjected wild type control.

Figure 5. p53 directly regulates the Δ113p53 expression. (A) PO construct as shown in Figure 3 with addition of showing the three predicted p53-binding sites in the regulatory regions I and Il in the Δ113p53 promoter. Site 1 :,

GGGCATGTTC (SEQ ID NO: 24); site 2:, TGACATGTTA (SEQ ID NO: 25); site

3:, GAACATGTCT (SEQ ID NO: 26). TSS: transcription start site. (B) Injecting def-MO alone (2^nd from the left) induced the Gfp fluorescence in the Tg(Δ113p53:gfp) transgenic embryos when compared with the uninjected control embryos (1^st from the left) or following co-injection of def-MO with either p53-MO^ATG (3^rd from the left) or p53-MO^spl (4^th from the left). (C) RNA gel blot hybridization showing the reduction in Gfp fluorescence in the def-MO and p53-

MO co-injected embryos due to reduced expression of gfp. 18S rRNA was used as the loading control. (D) The expression of HA-p53 protein in the injected embryos (input) and the effectiveness of chromatin immunoprecipitation (ChIP) of HA-p53 using the anti-HA antibody (α-HA pull down) examined by Western blotting. The uninjected embryos were used as the negative control. (E) ChIP products from different samples in (D) were used as the templates for PCRs using a pair of primers for exon 10 (negative control), a pair for region -112 to +98 (Site 3) and a pair for the region -1086 to -1300 (Site 1 ). Note the primer pairs for exon 10 (+1264 to +1480 in p53 cDNA with Accession Number AF365873) amplified a product only from the input sample (lower panel) but not the ChIP sample (upper panel) whilst both primer pairs for region -112 to +98 and region -1086 to -1300 yielded size-predicted products from the ChIP product.

Figure 6. Δ113p53 directly interacts with p53. (A) HΛ-tagged p53 (left) and MVC-tagged Δ113p53 (right) cloned into the expression vector pCS2+, respectively. (B) HA-p53 and MYC-Δ113p53 mRNAs were injected alone or co- injected into single-cell stage embryos and total proteins were extracted from embryos at 5 hours post-injection. Immunoprecipitation (IP) with anti-HA antibody precipitated HA-p53 and the IP products were detected for MYC- Δ113p53 using an anti-MYC antibody. The MVC-Δ113p53 protein was detected only in the IP products from embryos co-injected with HA-p53 and Δ113p53 mRNAs (top two panels). HA-p53 and /WYC-Δ113p53 proteins were expressed in the injected embryos (input, bottom two panels).

Figure 7. The expression of Δ113p53 is induced by γ-ray irradiation and drug treatments. (A) RNA gel blot hybridization using a p53 probe (top panel) or a

Δ113p53 specific probe (middle panel) showing that Δ113p53 expression was greatly induced in the embryos treated with either γ-ray or carcinogenic drugs camptothecin and rostovitine. 18S rRNA was used as the loading control. (B, C)

Both the Gfp fluorescence (B) and gfp transcripts (C) in the Tg(Δ113p53:gfp) transgenic embryos were also significantly induced by the same treatments. Figure 8. Knock-down Δ113p53 in zebrafish embryos caused high mortality. (A, B) Co-injection of Δ113p53-MO with the Δ113p53:gfp plasmid significantly decreased the levels of the Gfp protein (A) to cause weak Gfp fluorescence in the injected-embryos (B). In (A)₁ lane 1 , uninjected control; lane 2, Δ113p53:gfp plasmid injection; lane 3, Δ113p53:gfp plasmid + Δ113p53-MO co-injection. (C) Injection of Δ113p53-MO sensitized the WT embryos to γ-ray irradiation treatment and caused 100% mortality to the treated embryos (bottom right panel) at 5 days post-treatment. In contrast, the standard control morpholino injected embryos, though somehow deformed, exhibited ~50% surviving rate after the γ-ray irradiation treatment (top right panel) at 5 days post-treatment. The morpholino-injected γ-ray untreated controls are shown on the left.

Figure 9. Knock-down Δ113p53 caused massive cell apoptosis. Embryos 18 hpf post- γ-ray-treatment as in Figure 8 were used in TUNEL assay to detect cell apoptosis in each case.

Detailed description of the invention

Bibliographic references mentioned in the present specification are for convenience listed in the form of a list of references and added at the end of the examples. The whole content of such bibliographic references is herein incorporated by reference.

The present invention provides a transgenic non-human animal, wherein the transgenic non-human animal genome comprises at least one stably integrated reporter gene operably linked to regulatory elements of the p53 isoform gene, wherein the p53 isoform gene is capable of modulating the activity of p53. In particular, the reporter gene may be operably linked to the 5'- upstream region of a p53 isoform gene. The 5'-upstream of the p53 isoform gene may comprise the p53 isoform gene regulatory elements; however, the reporter gene may also be operably linked to other regulatory elements, for example enhancers, placed outside the 5'-upstream region of a p53 isoform gene. The p53 isoform gene may be Δ113p53 gene and the regulatory elements may be the Δ113p53 gene's regulatory elements. Further, the transgenic non-human animal may be a transgenic fish. In particular the non-human animal may be transgenic zebrafish.

The transgenic non-human animal according to the invention, may comprise at least one stably integrated reporter gene operably linked to a p53 isoform gene and/or its regulatory elements. In particular the reporter gene may be operably linked to a Δ113p53 gene isoform gene and/or its regulatory elements. The regulatory element may comprise a 4113bp 5'-upstream region, or portion thereof, of the translation start site of the p53 isoform gene. In particular, the regulatory element may comprise a 5'-upstream region -1041 to -1991bp, or portion thereof, of the transcription start site of the p53 isoform gene. More in particular, the regulatory element may comprise a 5'-upstream region -1 to -

239bp, or portion thereof, of the transcription start site of the p53 isoform gene.

For example, the regulatory element may comprise a promoter.

As defined herein, a nucleotide sequence is "operably linked" to another nucleotide sequence when it is placed in a functional relationship with another nucleotide sequence. For example, if a coding sequence is operably linked to a promoter sequence, this generally means that the promoter may promote transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary join two protein coding regions, contiguous and in reading frame. "Regulatory sequence" (also called regulatory region or ~ element) is a promoter, enhancer or other segment of DNA where regulatory proteins such as transcription factors bind preferentially. They control gene expression and thus protein expression. Regulatory sequences or elements can also be found in messenger RNA. They may be bound by RNA-binding proteins or RNAs (eg miRNAs). Since enhancers may function when separated from the promoter by several kilobases and intron sequences may be of variable lengths, some nucleotide sequences may be operably linked but not contiguous.

The transgene may be included in a vector for delivery. A vector, as used herein and as known in the art, refers to a nucleic acid construct that includes genetic material designed to direct transformation (i. e., the process whereby genetic material of an individual cell is altered by incorporation of exogenous DNA into its genome) of a targeted cell. A vector may contain multiple genetic elements positionally and sequentially oriented, i. e., operably linked with other necessary or desired elements such that the nucleic acid in a cassette can be transcribed and, if desired, translated in the microinjected, single-cell fertilized embryo.

Recombinant expression vectors may be constructed by incorporating the above-recited nucleotide sequences within a vector according to methods well known to the skilled artisan and as described, for example, in Sambrook and

Russell, 2001. A wide variety of vectors are known that have use in the invention. Suitable vectors include plasmid vectors, viral vectors, including retrovirus vectors. The vectors may include other known genetic elements necessary or desirable for efficient expression of the nucleic acid in a specified host cell, such as the transgenic fish host cells described herein, including regulatory elements.

A wide variety of fish may be utilized and the invention is in no way limited to the use of zebrafish. Exemplary fish include teleost fish, such as zebrafish (Danio rerio), medaka (Oryzas latipes), mummichog (Fundulus laeteroclitus), killifish (Genus Fudulus), catfish (Genus lctalurus), such as channel catfish; carp (Genus Cyprinus), such as common carp; and trout or salmon (e. g., Genus Salvelnus, Salvo, and Oncorhyflchus). Zebrafish, in particular, may be advantageously utilized as compared to other animal models. For example, zebrafish are amenable to genetic screens, modifier screens, and chemical screens; develop rapidly ex-utero; are transparent for much of their life cycle and produce large clutches of offspring weekly. Zebrafish can be raised in relatively small facilities (housing up to about 54 adult fish in a single 9 liter tank), and can reliably produce offspring in large quantities, with each mature female typically laying between 100 to 300 eggs per week. These eggs are fertilized externally, and the embryos are transparent allowing the early development of hematopoietic tissues and other organ and tissue systems to be directly observed using only a dissecting microscope. Embryonic development is extremely rapid with most organ systems including blood cell formation being fully developed by 5 days post fertilization. Full reproductive maturation is reached by about 3 months.

According to another aspect of the invention there is provided a method of detecting and/or monitoring nucleic acid damaging and/or carcinogenic agents in the environment comprising:

(a) contacting and/or exposing a transgenic non-human animal to the environment, wherein the transgenic non-human animal genome comprises at least one stably integrated reporter gene operably linked to a p53 isoform gene regulatory elements, wherein the p53 isoform gene is capable of modulating the expression and/or activity of p53; and

(b) determining the reporter gene expression, wherein the increased reporter gene expression compared to control reporter gene expression is indicative of the of presence of nucleic acid damaging and/or carcinogenic agents in the environment.

In particular, the p53 isoform gene may be Δ113p53 gene and the regulatory elements may be the p53 isoform gene's regulatory elements. The transgenic non-human animal may be a transgenic fish, in particular a transgenic zebrafish.

The reporter gene in the transgenic non-human animal according to the invention may be expressed at control expression levels. Accordingly an increased expression of the reporter gene, compared to the control expression level, may be induced by exposure of the non-human animal to nucleic acid damaging and/or carcinogenic agent(s). In particular an increased reporter gene expression, compared to the control expression level, is indicative of increase in the expression of the p53 isoform gene in at least one cell, wherein the increased expression of the p53 isoform gene prevents, reduces and/or inhibits the p53 activity in at least one cell. The increased expression of p53 isoform gene may further prevent apoptosis in at least one cell. The increased expression of p53 isoform gene in at least one cell may induce cancer progression.

The term "control expression levels" means that the expression of particular gene and/or mRNA transcript (for e.g., p53 isoform gene, in particular Δ113p53 gene or GFP) or the expression of a particular protein (p53 isoform, in particular Δ113p53 or GFP) is at a basal level of expression constitutively present in a cell in the absence of any stimulation. A fish may exhibit the control expression levels in an environment that is devoid of DNA modifying and/or carcinogenic agents. The term "carcinogenic agents" refers to any substance, radionuclide or radiation which is an agent directly involved in the promotion of cancer or in the facilitation of its propagation. This may be due to genomic instability or to the disruption of cellular metabolic processes. Carcinogenic agents may increase the risk of getting cancer by altering cellular metabolism or damaging DNA directly in cells, which interferes with biological processes, and induces the uncontrolled, malignant division by inhibiting the programmed cell death process of "apoptosis", ultimately leading to the formation and/or progression of cancer. DNA Modification occurs when the DNA is readily modified by substitution, deletion or insertion of nucleotides. Therefore, for ease of comprehension the carcinogenic agents are referred herein interchangeably as "DNA modifying agents". Accordingly, in the present invention an increased expression and/or activity of the reporter gene is in turn is indicative of an increased expression and/or activity of p53 isoform gene, more in particular of Δ113p53 gene. The term "increased expression and/or activity" as used herein means that the expression of particular gene and/or mRNA transcript or the expression of a particular protein is higher or elevated compared to the control expression or constitutive expression levels. Accordingly a fish may exhibit increased expression levels in the presence of DNA modifying and/or carcinogenic agents in the environment.

The reporter gene of the present invention may be selected from the group consisting of luciferase, galactosidase, chloramphenicol, acetyltransferase, b- glucuronidase, and alkaline phosphatase. The reporter gene may a fluorescent protein gene. The fluorescent protein may be selected from the group consisting of GFP, RFP, BFP, YFP, and dsRED2. In particular, the fluorescent protein may be GFP. The reporter gene may comprise a transcription stop-site.

A reporter gene (often simply reporter) as used herein is a gene that may be directly attached/fused to a promoter and/or may be attached/fused to another gene of interest in cell culture, animals and/or plants. The reporter gene in turn confers characteristics on organisms (e.g. on transgenic fish) expressing them so that they are easily identified and measured. The reporter gene may itself be placed in a DNA construct and inserted into the cell or organism. Alternatively, the reporter gene and the gene of interest (for example, the p53 isoform gene) may be placed in the same DNA construct and inserted into the cell or organism. Reporter genes that induce visually indentifiable characteristics usually involve fluorescent proteins; for example, the gene that encodes jellyfish green fluorescent protein (GFP)₁ which causes cells that express it to glow green under UV light. Other examples include the enzyme luciferase, which catalyzes a reaction with a luciferin to produce light, lacZ gene, which encodes the protein β-galactosidase that causes bacteria expressing the gene to appear blue when grown on a medium that contains the substrate analog X-gal (an inducer molecule such as IPTG is also needed under the native promoter). Reporters may also be selectable markers, for example the chloramphenicol acetyltransferase (CAT) gene, which confers resistance to the antibiotic chloramphenicol.

According to yet another aspect the present invention provides a method of screening drugs and/or agents capable of decreasing and/or inhibiting the expression of a p53 isoform gene, wherein the p53 isoform gene is capable of modulating the activity of p53, comprising (a) contacting and/or exposing a transgenic non-human animal to a test drug and/or agent, wherein the transgenic non-human animal genome has stably integrated a reporter gene operably linked to the p53 isoform gene regulatory elements; and (b) determining if the test drug and/or agent is capable of decreasing and/or inhibiting the expression of the p53 isoform gene. wherein the step b) comprises determining if the test drug and/or agent is capable of decreasing and/or inhibiting the expression of the reporter gene. In particular the p53 isoform gene may be Δ113p53 gene. The transgenic non- human animal is a transgenic fish. In particular the transgenic non-human animal is a transgenic zebrafish.

The reporter gene may selected from the group consisting of luciferase, galactosidase, chloramphenicol, acetyltransferase, b-glucuronidase, and alkaline phosphatase. In particular the reporter gene may a fluorescent protein gene, wherein the fluorescent protein is selected from the group consisting of GFP, RFP, BFP, YFP₁ and dsRED2. More in particular, the fluorescent protein may be GFP.

Drug candidates may be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity. The compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound" library method, and synthetic library methods using affinity chromatography selection. The biological library approach is typically used for polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds.

The test drug or agent is typically identified from a large-scale, robotically-d riven screen of thousands of compounds to identify a drug or agent thought to have the ability to modulate the expression of genes thought to be involved in cancer progression. In the present invention agents capable of modulating, particularly reducing and/or inhibiting, the expression of p53 isoform genes, in particular Δ113p53 may be selected. The drugs and/or agents may also modulate the sensitivity of transgenic cells to treatments with radiation or chemotherapy. Such screens are routine, and these, and other screening methods, are well known by those of skill in the art. The test drug and/or agent may reduce and/or inhibit, expression of p53 isoform genes, mRNA and/or protein and/or modulate other genes involved in cancer. In particular the test drug and/or agent may prevent, reduce and/or inhibit the expression of Δ113p53 gene, mRNA and/or protein and/or modulate other genes involved in cancer Additionally, the test drug and/or agent may inhibit or stimulate the activity of other molecules involved, directly or indirectly, or modulate the sensitivity of transgenic cells to treatments with radiation or chemotherapy. A wide variety of drugs and/or agents may be tested in the screening methods of the present invention. Small molecule compounds are identified by screening large chemical libraries for the effects of compound addition to the water of developing fish. Additionally, proteins such as oligo-and polypeptides, may also act as test drugs or agents.

Accordingly, the test drug and/or agent may be a nucleic acid molecule capable of binding and/or hybridising to the p53 isoform gene, mRNA, promoter and/or a portion thereof. The nucleic acid molecule may be complementary to and/or hybridise to the p53 isoform gene, mRNA, promoter and/or a portion thereof. In particular, the p53 isoform gene, mRNA, promoter and/or portion thereof may be Δ113p53 gene, mRNA, promotor and/or a portion thereof. Further the nucleic acid molecule may be an antisense nucleic acid molecule. The nucleic acid molecule may be an antisense single strand RNA (sRNA), double strand RNA (dsRNA), double strand DNA (dsDNA), double strand hybrid RNA/DNA (RNA/DNA), small interfering RNA (siRNA), micro RNA (miRNA), morpholinos (MO/PMO) and/or ribozymes. In particular, the nucleic acid molecule may be morpholino (MO/PMO).

The nucleic acid molecule of the invention may comprise a nucleotide sequence which is complementary to and/or which hybridizes to a 4113bp upstream region, or portion thereof, of the translation start site of the p53 isoform gene. In particular, the nucleic acid molecule may comprise a nucleotide sequence which is complementary to and/or which hybridizes to a 5'-upstream region - 1041 to -1991 bp or portion thereof of the transcription start site of the p53 isoform gene. More in particular, the nucleic acid molecule may comprise a nucleotide sequence which is complementary to and/or which hybridizes to a 5'- upstream region -1 to -239bp or portion thereof of the transcription start site of the p53 isoform gene.

The term "nucleic acid" is well known in the art and is used to generally refer to a molecule (one or more strands) of DNA, RNA or a derivative or analog thereof comprising nucleobases. A nucleobase includes, for example, a purine or pyrimidine base found in DNA (e.g., an adenine "A", a guanine "G", a thymine "T" or a cytosine "C") or RNA (e.g., an A, a G, an Uracil "U" or a C). The term nucleic acid encompasses the terms "oligonucleotide" and "polynucleotide" each as subgenus of the term "nucleic acid". The term "complementary" in the context of nucleic acids refers to a strand of nucleic acid non-covalently attached to another strand, wherein the complementarity of the two strands is defined by the complementarity of the bases. For example, the base A on one strand pairs with the base T or U on the other, and the base G on one strand pairs with the base C on the other. An oligonucleotide or analog is of "substantial complementarity" when there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide or analog to non-target sequences under conditions in which specific binding is desired

A nucleic acid molecule is "hybridisable" to another nucleic acid molecule (in the present case, the p53 isoform, for example Δ113p53 ), when a single-stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (Sambrook and Russell, 2001 ). The conditions of temperature and ionic strength determine the "stringency" of the hybridisation. Hybridisation requires the two nucleic acids to contain complementary sequences. Depending on the stringency of the hybridisation, mismatches between bases are possible. The appropriate stringency for hybridising nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridisation decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (Sambrook and Russell, 2001 ). For hybridisation with shorter nucleic acids, i.e. oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (Sambrook and Russell, 2001 ).

"Preventing, inhibiting and/or reducing the expression and/or activity" of a gene, mRNA and/or promoter used herein refers to the ability of the nucleic acid molecules described above, to measurably prevent, reduce and/or inhibit the expression and/or activity of a gene, mRNA and/or promoter. In the present invention it contemplates prevention, reduction and/or inhibition of the expression, activity and/or function of a particular gene and/or transcript. It is understood that the phrase is relative, and does not require absolute suppression of the transcript. Thus, in certain aspects, preventing, reducing and/or inhibiting the expression of p53 isoform gene and/or transcript, in particular preventing, reducing and/or inhibiting the expression of Δ113p53 gene and/or transcript, requires that, following application of the nucleic acid molecules mentioned in the previous section, p53 isoform gene and/or transcript, in particular Δ113p53 gene and/or transcript, is expressed at least 5 % less than prior to application these compounds and/or molecules, such as at least 10 % less, at least 15 % less, at least 20 % less, at least 25 % less, or even more reduced. Thus, in some particular aspects, application of the nucleic acid molecules reduces and/or inhibits expression of the the p53 isoform, in particular Δ113p53, by about 30 %, about 40 %, about 50 %, about 60 %, or more. In specific examples, where the nucleic acid molecules are particularly effective, expression is inhibited and/or reduced by 70 %, 85 %, 85 %, 90 %, 95 %, or even more. Anti-sense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule. In the cell, they hybridise to mRNA, forming an untranslatable double-stranded molecule. Therefore, antisense nucleic acids interfere with the expression of mRNA into protein. Oligomers of about fifteen nucleotides and molecules that hybridise to the AUG initiation codon will be particularly efficient, since they are easy to synthesise and are likely to pose fewer problems than larger molecules. Anti-sense methods have been used to inhibit the expression of many genes in vitro.

Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA molecules in a manner somewhat analogous to DNA restriction endonucleases. Ribozymes were discovered from the observation that certain mRNAs have the ability to excise their own introns. By modifying the nucleotide sequence of these RNAs, researchers have been able to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it. Because they are sequence-specific, only mRNAs with particular sequences are inactivated. For example, the nucleic acid molecule is an antisense DNA and/or RNA molecule. The nucleic acid molecule may be an antisense single strand RNA (sRNA), double strand RNA (dsRNA), double strand DNA (dsDNA), double strand hybrid RNA/DNA (RNA/DNA), small interfering RNA (siRNA) and/or ribozymes. The nucleic acid construct can be any suitable vector, phage, plasmid, a nucleic acid fragment or the like comprising the nucleic acid molecule.

According to a particular aspect, the inhibition, delay or repression is carried out by the silencing interference RNA (siRNA) technology. RNA interference technology is well known and consists of a process in which a double stranded RNA (dsRNA) induces the postranscriptional degradation of homologous transcripts. RNAi can be initiated by exposing cells to dsRNA either via transfection or endogenous expression. According to the exemplified embodiment, DNA targeting sequences, specific for the reduction and/or inhibition of the p53 isoform gene(s) and/or transcript, in particular reduction and/or inhibition of Δ133p53 or Δ113p53 gene and/or transcript, are selected and prepared according to standard technology, for example, the DNA targeting sequence are generated using Ambion siRNA target finder (http://www.ambion.com/techlib/misc/siRNA_finder.html). The DNA targeting sequences may be inserted into a construct and/or vector and used to transfect the cell or cell lines in vitro or in vivo. The RNA polymerase of the cell transcribes the siRNAs complementary to the p53 isoform transcript, in particular complementary to Δ133p53 or Δ113p53 transcript, or to a portion thereof. These siRNAs form a complex known as the RNA-induced silencing complex or RISC which functions in homologous target RNA destruction. In mammalian systems, the sequence-specific RNAi effect has been observed by the introduction of siRNAs either via transfection or endogenous expression of 19-23 base transcripts capable of forming duplexes, or via expression of short hairpin RNAs. The siRNA expression constructs and/or vectors may be constructed according to any method known in the art, for example by chemical synthesis, in vitro transcription, by digestion of long dsRNA by an RNase III family enzyme (e.g. Dicer, RNase III), by expression in cells from an siRNA expression plasmid or viral vector, and expression in cells from a PCR-derived siRNA expression cassette. The construct is directly transfected into mammalian cells resulting in functional expression of siRNAs.

The test drugs or agents are typically administered in an amount and for a time necessary to suppress, or otherwise alter, or enhance oncogene-mediated neoplastic or hyperplastic transformation. Such amounts and times may be determined by the skilled artisan by known standard procedures. Transgenic fish are typically contacted with the test drug or agent at a desired time after hatching. In other forms of the invention, the fish embryo contained with the fish egg may be contacted with the test drug or agent.

Determining if the test drug and/or agent prevents, reduces and/or inhibits p53 isoform genes, or otherwise modulates other genes may be performed by measuring the amount and/or size of tumors formed in the fish and/or measuring the rate of onset of tumor formation. Other indicators of modulation of the sensitivity of transgenic cells to treatments with radiation or chemotherapy, may also be measured. For example, when reporter gene fusion constructs are used, reporter gene expression may be determined using methods well known by those of skill in the art and as described herein. For instance, utilizing a tissue-specific promoter operably linked to a GFP-oncogene fusion construct will permit GFP fluorescence emitted from the protein specifically expressed in a particular tissue to be determined. Additional visual or other screens for metastatic tumors may also be used.

The present inventors here present data showing that zebrafish p53 isoform expression, in particular Ai13p53 expression, is significantly induced by radiation and drug treatments and is directly regulated by p53. In addition, Δ113p53 forms a complex with p53. More importantly, while most of the wild type control fish still could survive from γ-ray irradiation treatment, knock-down of Δ113p53 in irradiation-treated fish caused -100% mortality. This result demonstrates that Δ113p53 acts as a dominant negative regulator of p53 to protect cells from p53-induced and/or other factor-induced cell death. These results have immense implications in cancer treatment. In fact, cancer treatment coupled with knock-down of Δ113p53 becomes more efficient due to increased cancer cell death. In addition, because the Tg(A113p53:gfp) transgenic fish faithfuly recapitulate the expression regulation of the endogenous Δ113p53, the transgenic fish is also useful in screening for more effective drugs or factors for cancer treatment via chemical and/or genetic approaches. Further, the Tg(A113p53:gfp) transgenic fish may also be used as a convenient reporter system to monitor the environment pollutants.

Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention.

EXAMPLES

Standard molecular biology techniques known in the art and not specifically described were generally followed as described in Sambrook and Russel, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2001 ).

Materials and methods

Δ113p53:gfp plasmid

A 4.113 kb DNA fragment immediately upstream of the start codon ATG of Δ113p53 was amplified from genomic DNA (AB strain wild type zebrafish) with primer pair PO-F and PO-R (SEQ ID NO: 1 and SEQ ID NO: 2) (Table 1 ) using the Expand Long Template Kit (Roche). Both the primers were derived from the p53 mRNA (accession number AF365873). The PCR product was first cloned into pGEMTeasy vector (Promega), then digested with BamHI and EcoRI enzymes before being subcloned into the pEGFP-1 vector (Clontech) (T4 ligase used was from Promega) to generate the Δ113p53:gfp plasmid.

Table 1 : Primers Used for Various Characterization and Cloning Purposes

Primers for promoter deletion constructs SEQ ID NOs

P1 pΔ113p53-(-2543U)-ιnfusιon; 5'CTCAAGCTTCGAATTAGTAAAGGGACAAGCTGTTTA

PZ pΔ113p53-(-1991 U)-ιnfusιon; δ'CTCAAGCTTCGAATTACAGCGTACCACCCACAACCA

LacZ-low: 5'-ATCGTAACCGTGCATCTG-S' 38

Primers for Def

Def-up: S'-TATTGCCTTACGACAGTTT-S' 39

Def-low: S'-CAAGCGTTTGACATTAGAGT-S' 40

Table 2. The p53 probe I that can detect both p53 and A113p53 transcripts (SEQ ID NO: 35) atggcgcaaaacgacagccaagagttcgcggagctctgggagaagaatttgattattcagcccccaggtggtggctcttg ctgggacatcattaatgatgaggagtacttgccgggatcgtttgaccccaatttttttgaaaatgtgcttgaagaacagc ctcagccatccactctcccaccaacatccactgttccggagacaagcgactatcccggcgatcatggatttaggctcagg ttcccgcagtctggcacagcaaaatctgtaacttgcacttattcaccggacctgaataaactcttctgtcagctggcaaa aacttgccccgttcaaatggtggtggacgttgcccctccacagggctccgtggttcgagccactgccatctataagaagt ccgagcatgtggccgaagtggtccgcagatgcccccatcatgagcgaaccccggatggagataacttggcgcctgctggc catttgataagagtggagggcaatcagcgagcaaattacagggaagataacatcactttaaggcatagtgtttttgtccc atatgaagcaccacagcttggtgctgaatggacaactgtgctactaaactacatgtgcaatagcagctgcatggggggga tgaaccgcaggcccatcctcacaatcatcactctggagactcaggaaggtcagttgctgggccggaggtcttttgaggtg cgtgtgtgtgcatgtccaggcagagacaggaaaactgaggagagcaacttcaagaaagaccaagagaccaaaaccatggc caaaaccaccactgggaccaaacgtaaatcttcttcagctacatcacgacctgaggggagcaaaaaggccaagggctcca gcagcgatgaggagatctttaccctgcaggtgaggggccgggagcgttatgaaattttaaagaaattgaacgacagtctg gagttaagtgatgtggtgcctgcctcagatgctgaaaagtatcgtcagaaattcatgacaaaaaacaaaaaagagaatcg

Table 3. The A113p53 specific probe derived the 5' UTR of A113p53 (SEQ ID NO:36) cgcatttttaaaatatcctggcgaacatttggagggagatgttggtctttttatgcattttttaggatggagtgtaatacattttagg attgttaatagtgctggacagtcaagctggtgcttcacattctgtgtgacattacaagaccaggagg

Tg(Δ113p53:gfp) transgenic fish The Δ113p53:gfp plasmid used for injection was linearized with EcoRI. The Δ113p53:gfp plasmid was linearized and injected into single-cell stage embryos. Approximately 25 pg of linearized Δ113p53:gfp plasmid DNA was injected into the one-cell stage embryos thereby generating TO stage fish. The TO fish were raised to adulthood for screening for individual fish that produced Gfp positive offspring. Total three independent TO lines were found to produce gfp positive transgenic fish (Tg(Δ113p53:gfp) transgenic fish). These TO fish were then mated with the wild type (WT) fish to generate the T1 progenies. The progenies T1 from each individual TO x WT were raised to adulthood and then used in screening for gfp positive fish using gfp specific primers EGFP-U322 and EGFP-L742 (SEQ ID NO: 14 and SEQ ID NO: 15)(Table 1 ). Homozygous Tg(Δ113p53:gfp fish were obtained by mating a male and female gfp positive T1 fish in the same family. Homozygous Tg(Δ113p53:gfp fish were mated with d_ef^hι429 heterozygotes (Chen et al., 2005) to generate the F1 population and F1 individuals heterozygous both for gfp and def"⁴²⁹ were identified and mated to generate the F2 population. The Gfp fluorescence in the F2 population was examined under a Leica DMIRE2 fluorescence microscope and Gfp positive embryos were genotyped for the def"⁴²⁹ mutation using the lacz and def gene specific primers (SEQ ID NOS: lacz: 37 AND 38; def. 39 and 40)(Table 1 ).

Deletion analysis of the Δ113p53 promoter region Total nine different deletion constructs were made from the initial 4.113 kb DNA fragment (Figure 3). Each truncated promoter was amplified from Δ113p53:gfp (PO) using iProof enzyme (Biorad) and cloned into pEGFP-1 vector using the Infusion kit (Clontech) according to manufacturer's protocol. Corresponding primer pairs used are listed in Table 1. The PO construct was made using primers comprising the sequence of SEQ ID NO: 1 and SEQ ID NO: 2. The P1 , P2, P3, P4 and P5 constructs were made using the sequences comprising the SEQ ID NOs: 3, 4, 5, 6 and 7 respectively as forward primers. The primer comprising the sequence of SEQ ID NO: 2 was used as reverse primer in the constructs P1 to P5. The internal deletion constructs without the nucleotides (- 1059) to (-506) designated construct P6 or the deletion construct without the nucleotides (-239) to (-1 ) designated construct P9 were also derived from Δ113p53:gfp using iProof enzyme (Biorad) (Figure 3). To make the P6 construct, primer pairs (-U3059)-(-L1060) (SEQ ID NO: 1 and SEQ ID NO: 9) and (-U505ML1054) (SEQ ID NO: 8 and SEQ ID NO: 2) were used to amplify the parts flanking the left and right side of the deletion, respectively (Figure 3). PCR products from these two pairs of primers were denatured and mixed together to allow annealing of the sticky ends and this mixture was then used as the templates for the second round PCR using primers (-U3059) and (L1054) (SEQ ID NO: 1 and SEQ ID NO: 2) to get the product P6 with the internal deletion of the nucleotides -1059 to -506. Similarly, for P9, primer pairs (- U3059)-( -L240) (SEQ ID NO: 1 and SEQ ID NO: 13) and (U1 )-(L1054) (SEQ ID NO: 12 and SEQ ID NO: 2) were used to amplify the two parts flanking each side of the deletion and the PCR products were mixed together and used as the template for the second round PCR using primers (-U3059) and (L1054) (SEQ ID NO: 1 and SEQ ID NO: 2) to get the internal deletion product for P9. The final PCR products were cloned into pEGFP-1 to get P6 and P9 plasmids, respectively, using the Infusion kit (Clontech) according to manufacturer's protocol. All fragments cloned were sequenced and confirmed to be identical to Δ113p53:gfp except the deleted regions. Ten pg of each plasmid DNA was injected into AB fish embryos at one-cell stage.

Chromatin immunoprecipitation (ChIP)

Approximately 200 pg of HA taggedp53 mRNA was injected into one-cell stage embryos. At 5 hours post fertilization (hpf), -500 embryos were deyolked in PBS with 1x protease inhibitor cocktail (Complete, Roche). The supernatant was removed after centrifugation at 300 g. The pellet was homogenized in 1 ml NIM buffer of 0.25mM sucrose, 25 mM KCI, 10 mM Tris. Cl (pH 7.4), 5mM MgCI2 and ixComplete and treated with formaldehyde (final concentration 1 %) at room temperature for 15 minutes. The reaction was quenched with glycine (final concentration 125mM). The suspension was pelleted at 80Og in 4⁰C. The pellet was washed with NIM buffer three times, and resuspended in SDS lysis buffer (provided with ChIP assay kit; Upstate Biotechnology). The embryo lysate was sonicated to shear the chromatin by subjecting the lysate to 40 sets of 5- second pulses using Misonix 3000 equipped with a 2-mm tip and the energy output was set to 2. The lysate was incubated on ice for 2 min between each pulse. The chromatin was sheared into 200-1000 bp fragments. After sonication, 50 ul lysate was taken out as the template for positive control PCRs and 25 ul lysate for western blot. The rest of lysate was spun at 14,000 rpm and the pellet was resuspended in ChIP dilution buffer. 100 ul HA antibody-matrix (Roche) was added to the suspension and incubated overnight at 4⁰C. Following the incubation the suspension was washed and the HA antibody- matrix was eluted as described in the protocol (given by the ChIP assay kit). The histone-DNA crosslinks was reversed according to manufacture's protocol. The DNA was recovered by phenol/chloroform extraction and precipitated by ethanol. The pellet was resuspended in distilled water and used as the template for PCR reactions.

Co-immunoprecipitation (Co-IP) analysis

The entire coding sequences of p53 and Δ113p53 were respectively amplified with corresponding primer pairs BamHI-HA-fullATG (SEQ ID NO: 21 ) and EcoRI-tp53r (SEQ ID NO: 23) or BamHI-myC-113ATG (SEQ ID NO: 22) and EcoRI-tp53r (SEQ ID NO: 23) (Table 1) using the Phusion enzyme (ATI). PCR products were digested with BamHI and EcoRI and each amplicon was respectively ligated into pCS2+ vector using Rapid Ligation Kit (Roche) to generate two pCS2+ constructs. To facilitate our studies p53 was fused with a HA-tag at its N-terminal in one construct (pCS2+-HA-p53) and Δ113p53 fused to a MYC-tag at its N-terminal in the other construct (pCS2+-MYC- Δ113p53)(F\gure 6A).

The pCS2+-HA-p53 and pCS2+-MYC-Δ113p53 plasmids were linearized with Not1 for synthesizing the HA-p53 and MYC-Δ113p53 mRNA with the mMESSAGE mMACHINE^® SP6 kit (Ambion), respectively. Approximately 200 pg of HA-p53 and 500 pg of MYC-Δ113p53 mRNA was either injected separately, or co-injected into WT embryos (AB strain) at one-cell stage. The un-injected embryos were used as the control. At 5 hours post-injection, -200 embryos of each injection were deyolked as mentioned above. The pellet was homogenized in Tris.CI Lysis Buffer of 50 mM Tris.CI pH 8.0, 150 mM NaCI, 0.1% NP40, 0.1 mM DTT and 1x Complete. The lysate was centrifuged at 14,000 rpm for 5 min at 4⁰C. Before being loaded onto the CO-IP column (PIERCE), 20 ul supernatant was taken out as the input control. The rest was mixed with 6 ul Anti-HA agarose. The subsequent procedures were according to the manufacture's instructions (PIERCE). All samples were equally loaded into two PAGE gels for western blot analysis, one was probed with an anti-HA antibody and the other with an anti-MYC antibody.

Morpholinos

All morpholinos were purchased from Gene Tools. Two p53 antisense morpholinos, one targeting against the start codon ATG comprising the sequence of SEQ ID NO: 16 (δ'-GCGCCATTGCTTTGCAAGAATTG-S¹) (Langheinrich et al., 2002) (p53-MO^ATG) and the other against the splice junction between exon 5 and intron 5 (δ'-AAAATGTCTGTACTATCTCCATCCG- (p53-MO^spl) comprising the sequence of SEQ ID NO: 17 (Chen et al., 2005), were designed. Further def antisense morpholino corresponding to the splice junction between exon 2 and intron 2 and comprising the sequence of SEQ ID NO: 18 (5'-ATGAATATAATGACTTACCAAGCGC-S') (def-MO) was also designed (Chen et al., 2005) The morpholinos were injected at a concentration was 1.0 mM. An antisense morpholino comprising the sequence of SEQ ID NO: 19 (5'-GCAAGTTTTTGCCTGACAGAAG-S') (Δ113p53-MO) that specifically targets against the 5'-UTR region of Δ113p53 to block its translation was designed. The human beta-globin antisense morpholino comprising the sequence of SEQ ID NO: 20 (δ'-CCTCTTACCTCAGTTACAATTT-S') was used as the standard control (st-MO). The two later morpholinos were used in injection at a concentration of 0.4 mM.

γ-ray irradiation and drug treatment Embryos at 1 day(s) post fertilization (dpf) were γ-ray irradiated (with a dosage of 24 Gray) or treated with 500 nM campthecin or 50 um roscovitine, respectively. At 18 hours post-treatment, the embryos were harvested for RNA extraction and the extracted RNA was used for Northern blot analysis of p53 and Δ113p53 expression. To assay the response of the Δ113p53 promoter, heterozygous Tg(Δ113p53:gfp) transgenic embryos were treated with γ-ray and the two drugs, respectively. RNA was extracted from the treated embryos and used for the analysis of gfp transcripts.

Cell apoptosis assay and mortality counting: 0.4 mM Δ113p53-MO or human beta-globin antisense morpholino (st-MO) or phenol red dye was respectively injected into one-cell stage embryos. At 1 dpf, embryos were γ-ray- irradiated with a dosage of 24 Gray. Embryos 18 hours post-γ-ray-irradiation were fixed with 4% paraformaldehyde (PFA) overnight and then subjected to the TUNEL assay using the In Situ Cell Death Detection Kit, TMR red (Roche) (Chen et al., 2005). At 5 days post-γ-ray-irradiation, the mortality for each treatment was counted.

Results

Identification of two regulatory c/s-regions essential for A113p53 expression

We have previously shown that the expression of Ai13p53 and some p53- response genes were strikingly up-regulated in the def ^hl429 mutant (Chen et al., 2005). In the current study injection of plasmid DNA induced the expression of Δ113p53 in zebrafish embryos (Figure 2A). To find out how A113p53 expression is regulated, a 4.113 kb DNA fragment immediately upstream the start codon ATG (containing part of exon 1 , exons 2-4, introns 1-4 and part of exon 5 of zebrafish p53) (Figure 1 ) was amplified using primers PO-F and PO-R (SEQ ID NO: 1 and SEQ ID NO: 2) and then cloned to the pEGFP vector upstream of the GFP reporter gene (A113p53:gfp). Because PO-F SEQ ID NO: 1 (starts from +26) and PO-R SEQ ID NO: 2 (ends at +477) are derived from the p53 mRNA (accession number AF365873) this DNA fragment excludes the p53 promoter region. Because the endogenous A113p53 expression is induced by plasmid DNA injection (Figure 2A) it is expected that injection of the A113p53:gfp plasmid would induce the gfp expression if the 4.113 kb DNA fragment contains the regulatory elements for the expression of A113p53. Therefore injection of the A113p53:gfp plasmid would also induce the gfp expression. Indeed, when the pA113p53::gfp plasmid was injected into the single-cell stage embryos gfp was strongly expressed in the injected embryos (Figure 2B), suggesting the presence of a functional promoter capable of driving the expression of A113p53 gene. We then generated Tg(Δ113p53:gfp) transgenic fish as described in materials and methods by injecting the A113p53:gfp plasmid DNA to the single-cell stage embryos and finally we obtained the Tg(Δ113p53:gfp) homozygous fish. The Tg(Δ113p53:gfp) homozygous fish was crossed with the def ^hl429 heterozygotes and the F2 progeny was checked for Gfp fluorescence in digestive organs. In F2 fish, at 1 dpf and 2 dpf, only weak gfp expression was detected in the wild type (WT) and in the def¹'⁴²⁹ heterozygous siblings (sb) but Gfp was strongly and ubiquitously expressed in the head region but relatively weakly expressed in the trunk region in the def¹'⁴²⁹ homozygous mutant (def-/-) (Figure 2C). At 3 dpf, Gfp continued to be weakly expressed in the WT and def"⁴²⁹ heterozygous siblings (sb) but is highly enriched in the head region and digestive organs in the def¹'⁴²⁹ homozygous mutants (def-/-), displaying a pattern similar to the endogenous A113p53 expression as reported previously (Figure 2D and 2E) (Chen et al., 2005). Therefore, these results indicate that 4.113 kb DNA fragment contained necessary regulatory elements for A113p53 expression in response both to stress signals (e.g plasmid DNA injection) and developmental cues (e.g the def"⁴²⁹ mutation). Because the PO-R (SEQ ID NO:2) primer is immediately upstream of the start codon ATG of A113p53 it indicated that the amplified 4.113 kb fragment contained the 5'-untranslated region (5'-UTR) of A113p53. Indeed, sequencing the 5'-race product via using a gfp specific primers (SEQ ID NO: 14 and SEQ ID NO: 15) revealed that the gfp transcript contained the 5'- UTR region just ahead of the start codon ATG of A113p53.

To further characterize the A113p53 promoter in detail, a series of deletion constructs in pEGFP-1 were prepared (Figure 3) and assayed for their ability to respond to stress signals in wild type embryos. Both the Gfp fluorescence (Figure 4A) and gfp transcripts together with the endogenous A113p53 transcripts (Figure 4B) were examined in each case. Deletion from -1992 to - 3059 from the 5'-end (construct P2, Figure 3) did not alter the promoter activity in driving the expression of gfp report gene (Figure 4A and 4B), however, deletion from -1544 to -3059 from the 5'-end (construct P3, Figure 3) significantly reduced the promoter activity in driving the expression of the gfp report gene (Figure 4A and 4B), suggesting the presence of a cis-regulatory element(s) in the region between -1544 to -1991 bp. Extension of deletion from -1544 to -1041 bp (construct P4, Figure 3) further reduced the promoter activity but this reduction was not enhanced by further deletion up to -506 bp (construct 5, Figure 3; Figure 4A and 4B), suggesting that there might be (an) additional regulatory element(s) between - 1041 and -1543 bp but not between -506 and - 1040 bp. This conclusion is confirmed by the observation that an internal deletion from -506 and -1059 did not alter the promoter activity (construct P6, Figure 3; Figure 4A and 4B). Deletion +564 to +1054 bp from the 3' end (construct P8, Figure 3) did not alter the promoter activity because the gfp transcript levels were similar to that observed for the control (PO construct) (Figure 4B). However, the Gfp fluorescence level was greatly reduced in P8 construct, (Figure 4A) and this can be explained by the reason that the P8 construct probably lacks a suitable Kozak sequence for efficient translation. On the other hand, deletion -278 to +1054 bp from the 3'-end (construct P7, Figure 3) completely abolished the promoter activity (Figure 4A and 4B) most likely because the -278 to +1054 bp region contains (a) crucial regulatory element(s) together with the transcription initiation start site (TSS, position +1 , Figure 3). To find out if there is(are) regulatory element(s) in the vicinity of TSS, we deleted the internal region between -1 to -239 bp upstream of the TSS (construct P9, Figure 3) and found this internal deletion also completely abolished the expression of gfp (Figure 4A and 4B). Therefore, this region is also crucially important for the A113p53 promoter activity. In conclusion, deletion analyses have identified two regions, namely -1041 to -1991 bp (region I) and -1 to -239 bp (region II) to contain crucial c/s-elements for the A113p53 expression. RT- PCR results showed that, in the all above cases, the endogenous A113p53 expression was increased due to stress signals (plasmid DNA injection) (Figure 4B), demonstrating that the two regions identified in our promoter analysis are likely genuinely used to regulate A113p53 expression in vivo.

A113p53 expression is directly regulated by p53

Sequence analysis showed that both regions I and Il contained putative p53 biding sites (one binding site around -1115 bp in region I, two binding sites between -94 and -242 bp in region II; Figure 5A), indicating that the expression of A113p53 might be p53-dependent. To study if A113p53 expression is regulated by p53, we co-injected either of the two p53 antisense morpholinos, one targeting the p53 start codon ATG to block the translation of p53 protein (p53-MO^ATG, SEQ ID NO: 16) and the other specifically targeting the splicing site of exon 5 and intron 5 of the p53 transcript (p53-MO^spl, SEQ ID NO: 17), which can efficiently knock-down both p53 and A113p53 expression; (Chen et al., 2005), with a def specific antisense morpholino (def-MO, SEQ ID NO: 18) into the Tg(Δ113p53:gfp) transgenic embryos. Uninjected embryos were used as the negative control and def-MO along injected embryos were used as the positive control. It has been previously shown that injection of the def-MO caused an upregulation of the A113p53 expression as that observed in the def¹'⁴²⁹ mutant. As expected, injection of def-MO alone caused obvious upregulation of gfp expression in the Tg(Δ113p53:gfp) embryos. In contrast, the Δ113p53 expression induced by the def-MO was significantly downregulated by co-injection of p53-MO^ATG and even more dramatically downregulated by p53- MO^spl suggesting that the p53 morpholinos compromised the effect of def-MO on inducing Gfp fluorescence (Figure 5B and 5C) and further demonstrating that A113p53 expression is p53-dependent.

To examine if p53 binds to the predicted putative p53 binding sites within the promoter of A113p53 so that to regulate A113p53 expression directly, HA- tagged p53 mRNA was injected into single-cell-stage embryos. The embryos at 5 hours post-injection were harvested and used for the chromatin co- immunoprecipitation (ChIP) assay using anti-HA antibody to pull down the HA- p53-DNA complex. Two pairs of primers, one pair CHIP-(-U1300) and CHIP- (L1086) (SEQ ID NOs: 29 and SEQ ID NO: 30) to amply the -1086 to -1300 fragment containing the putative p53-binding site in region I and the other pair CHIP-(-U112) and CHIP-(L98) (SEQ ID NO:31 and SEQ ID NO: 32) to amplify the -112 to +98 fragment containing one putative p53-binding site in region II, were designed and used to perform PCR using the HA-p53-DNA complex as the template. The results showed that both the -112 to +98 fragment and -1086 to -1300 fragment were specifically co-immunoprecipitated together with HA- p53 (Figure 5D and 5E). On the other hand, PCR using a pair of primers CHIP- EXON10-U and CHIP-EXON10-L (SEQ ID NO: 33 and SEQ ID NO: 34) derived from the exon 10 of p53 failed to yield any products from using the HA-p53-DNA complex as the template (Figure 5D). Therefore, these results indicate that p53 directly regulates A113p53 expression.

Δi13p53 forms a complex with p53

Both ΔNp63 and ΔNp73 are found to function as dominant negative regulators of p63 and p73, respectively, although they alone also have some biological functions (Grab et al., 2001 ; Yang et al., 1998). Similarly, Δ133p53 has been implicated to act as a dominant negative regulator of p53 since co-transfection p53 with Δ133p53 impaired p53-induced cell apoptosis (Bourdon et al., 2005). The Δ113p53 protein is an N-terminal truncated form of p53 with deletion of the activation domain and Mdm2-interacting motif and with partial deletion of the DNA-binding domain but retains the dimerization domain. To confirm that Δ113p53 acts as a dominant negative regulator of p53 and that Δ113p53 competes with p53 to form a heterodimer with p53, we investigated if p53 and Δ113p53 interacted with each other to facilitate Δi 13p53's dominant negative role on p53. The HA-tagged-p53 and MYC-tagged-A113p53 were cloned into pCS2+ expression plasmid, respectively (Figure 6A). The HA-tagged-p53 and MYC-tagged-A113p53 mRNA was obtained from in vitro transcription from corresponding plasmids and was co-injected into the single-cell stage zebrafish embryos. Injected embryos at 5 hpf were harvested and total proteins were extracted from the injected experimental and control embryos, respectively. HA- p53 was immunoprecipitated with anti-HA antibody and the protein precipitates were assayed for Δ113p53 using anti-MYC antibody. The results showed that Δ113p53 was efficiently co-precipitated with p53 (Figure 6B), demonstrating that p53 and Δ113p53 can form a complex.

γ-ray irradiation and drug treatments induce A113p53 expression

We have shown that injection of plasmid DNA induced the expression of A113p53 in the injected embryos (Figure 2A). This indicated that the injected plasmid DNA mimicked genomic DNA with broken ends that in turn triggered the cellular response. If this is the case, other means of causing DNA damage, such as γ-ray irradiation and drug treatment, would be expected to trigger the expression of A113p53 as well. The WT fish embryos were treated with γ-ray, camptothecin and rostovitine, respectively, and embryos 18 hours post- treatment were harvested and assayed for A113p53 expression using either a p53 probe (Table 2, Chen et al., 2005) (SEQ ID NO: 35) (which can detect both p53 and A113p53) or a A113p53 specific probe (Table 3, Cheng et al., 2005) (SEQ ID NO: 36). The results showed that all treatments dramatically induced the A113p53 expression in the treated embryos (Figure 7A). We also treated the Tg(Δ113p53:gfp) transgenic embryos with γ-ray, camptothecin and rostovitine and assayed both Gfp fluorescence and gfp transcripts in the treated embryos. The result showed that both levels of Gfp fluorescence and gfp transcripts were induced by these treatments (Figure 7B and 7C) and the γ-ray showed much stronger effect than the two drugs on induction of gfp expression (Figure 7C). This data further demonstrates that the 4.113 kb promoter region of A113p53 can faithfully recapitulate its regulation on endogenous A113p53 expression we analyzed so far.

Knock-down Δ113p53 in γ-ray-treated embryos caused high mortality

If Δ113p53 indeed functions as a dominant negative regulator of p53 as proposed, the fact that the A113p53 expression is greatly induced by stress signals such as γ-ray irradiation, injection of plasmid DNA and drug treatment suggests that Δ113p53 might be essential for cell survival by helping cells escaping from p53-induced cell apoptosis under these stress conditions. To test this hypothesis, we then investigated if reducing Δ113p53 via morpholino- mediated gene knock-down in the γ-ray irradiation treated embryos has any effects on embryo development. We first designed a morpholino (Δ113p53-MO) derived from the 5'-UTR of Ai13p53 (SEQ ID NO: 19) (this 5'-UTR region is included in the 4.113 kb pA113p53 fragment and is transcribed together with gfp in A113p53:gfp) and then co-injected Δ113p53-MO with Ai13p53:gfp plasmid into the single-cell stage embryos. We found that Δ113p53-MO can effectively knock-down the expression of Gfp protein (Figure 8A and 8B). When the Δ113p53-MO-injected embryos were treated with γ-ray (24 Gray) we found that the Δ113p53-MO injected embryos exhibited 100% mortality at 5 days post- treatment (448 embryos examined). On the other hand, both phenored dye and standard control morpholino (st-MO) injected wild type embryos could survive against 24 Gray dosage treatments (Figure 8C) and exhibited 67.4% and 51.5% surviving rate, respectively (more than 200 embryos examined in each case). TUNEL assay showed that the cause of the high mortality by γ-ray irradiation was likely due to the drastic increase of apoptosis in the Δ113p53-MO injected embryo (Figure 9; 18 hours post-treatment). Therefore, the normal function of Δ133p53/Δ113p53 is to protect cell survival against DNA-damaging stress environment.

References Bourdon, J-C, Femandes, K., Murray-Zmijewski, F., Liu, G., Xirodimas, D. P., Saville, M. K., and Lane, DP. 2005. p53 isoforms can regulate p53 transcriptional activity. Genes Devevelopment 19: 2122-2137.

Chen, J., Ruan, H., Ng, S.M., Gao, C, Soo, H. M., Wu, W., Zhang, Z., Wen, Z., Lane, D. P., and Peng, J. R. 2005. Loss of function of def selectively up-regulates Δi 13p53 expression to arrest expansion growth of digestive organs in zebrafish. Genes Development 19: 2900-2911.

Grab, TJ. , Novak, U., Maisse, C, Barcaroli, D., Lϋthi, A.U., Pirnia, F., Hϋgli, B., Graber, H. U., De Laurenzi, V., Fey, M. F., Melino, G. and Tobler, A. 2001. Human ΔNp73 regulates a dominant negative feedback loop for Tap73 and p53. Cell Death and Differentiation 8: 1213-1223.

Langheinrich, U., Hennen, E., Stott, G., and Vacun, G. 2002. Zebrafish as a model organism for the identification and characterization of drugs and genes affecting p53 signaling. Current Biology 12: 2023-2028.

Sambrook and Russell; 2001. Molecular cloning: A Laboratory manual, Cold Spring Harbour Laboratory press, New York.

Yang, A., Kaghad, M., Wang, Y., Gillet, E., Fleming, M., Dotsch, V., Andrews, N. C, Caput, D., and Mckeon, F. 1998. p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. Molecular Cell 2: 305-316.

Claims

1. A transgenic non-human animal, wherein the transgenic non-human animal genome comprises at least one stably integrated reporter gene operably linked to regulatory elements of a p53 isoform gene , wherein the p53 isoform gene is capable of modulating the activity of p53.

2. The transgenic non-human animal according to claim 1 , wherein the p53 isoform gene is Δ113p53 gene and the regulatory elements are the Δ113p53 gene's transcriptional regulatory elements.

3. The transgenic non-human animal according to any one of the preceding claims, wherein the transgenic non-human animal is a transgenic fish.

4. The transgenic non-human animal according to any one of the preceding, wherein the transgenic non-human animal is a transgenic zebrafish.

5. The transgenic non-human animal according to any one of the preceding claims, wherein the regulatory element comprises a 4113bp

5'-upstream region, or portion thereof, of the translation start site of the p53 isoform gene.

6. The transgenic non-human animal according any one of the preceding claims, wherein the regulatory element comprise a 5'-upstream region -

1041 to -1991 bp, or portion thereof, of the transcription start site of the p53 isoform gene.

7. The transgenic non-human animal according to any one of the preceding claims, wherein the regulatory element comprise a 5'- upstream region -1 to -239bp, or portion thereof, of the transcription start site of the p53 isoform gene.

8. The transgenic non-human animal according to any of the preceding claims, wherein regulatory element comprises a promoter.

9. The transgenic non-human animal according to any of the preceding claims, wherein the reporter gene is expressed at a control expression levels.

10. The transgenic non-human animal according to any of the preceding claims, wherein an increased expression of the reporter gene, compared to the control expression level, is induced by exposure of the non-human animal to nucleic acid damaging and/or carcinogenic agent(s).

11. The transgenic non-human animal according to any one of the preceding claims, wherein an increased reporter gene expression, compared to the control expression level, is indicative of increase in the expression of the p53 isoform gene in at least one cell.

12. The transgenic non-human animal according any one of the preceding claims, wherein increased expression of the p53 isoform gene prevents, reduces and/or inhibits the p53 activity in at least one cell.

13. The transgenic non-human animal according any one of the preceding claims, wherein increased expression of p53 isoform gene prevents apoptosis in at least one cell.

14. The transgenic non-human animal according to any of the preceding claims, wherein the increased expression of p53 isoform gene in at least one cell induces cancer progression.

15. The transgenic non-human animal according to any one of the preceding claims, wherein the reporter gene is selected from the group consisting of luciferase, galactosidase, chloramphenicol, acetyltransferase, b-glucuronidase, and alkaline phosphatase.

16. The transgenic non-human animal according to any one of the preceding claims, wherein the reporter gene is a fluorescent protein gene.

17. The transgenic non-human animal according to claim 16, wherein the fluorescent protein is selected from the group consisting of GFP, RFP, BFP, YFP, and dsRED2.

18. The transgenic non-human animal according to claim 16, wherein the fluorescent protein is GFP.

19. The transgenic non-human animal according to any of the preceding claims, wherein the reporter gene comprises a transcription stop-site.

20. A method of detecting and/or monitoring nucleic acid damaging and/or carcinogenic agents in the environment comprising: a) contacting and/or exposing a transgenic non-human animal to the environment, wherein the transgenic non-human animal genome comprises at least one stably integrated reporter gene operably linked to p53 isoform gene regulatory elements, wherein the p53 gene is capable of modulating the expression and/or activity of p53; and b) determining the reporter gene expression, wherein the increased reporter gene expression compared to control reporter gene expression is indicative of the of presence of nucleic acid damaging and/or carcinogenic agents in the environment.

21. The method according to claim 20, wherein the p53 isoform gene is Δ113p53 gene.

22. The method according to claim 20 or 21 , wherein the transgenic non- human animal is a transgenic fish.

23. The method according to any one of claims 20 to 22, wherein the transgenic non-human animal is a transgenic zebrafish.

24. The method according to any one of claims 20 to 23, wherein the reporter gene is selected from the group consisting of luciferase, galactosidase, chloramphenicol, acetyltransferase, b-glucuronidase, and alkaline phosphatase.

25. The method according to any one of claims 20 to 24, wherein the reporter gene is a fluorescent protein gene.

26. The method according to claim 25, wherein the fluorescent protein is selected from the group consisting of GFP, RFP, BFP, YFP, and dsRED2.

27. The method according to claim 25, wherein the fluorescent protein is GFP.

28. A method of screening drugs and/or agents capable of decreasing and/or inhibiting the expression of a p53 isoform gene, wherein the p53 isoform gene is capable of modulating the activity of p53, comprising a) contacting and/or exposing a transgenic non-human animal to a test drug and/or agent, wherein the transgenic non-human animal genome has stably integrated a reporter gene operably linked to the p53 isoform gene and/or its regulatory elements; and b) determining if the test drug and/or agent is capable of decreasing and/or inhibiting the expression of the p53 isoform gene.

29. The method of screening according to claim 28, wherein the screening is in the presence of nucleic acid damaging and/or carcinogenic agent(s).

30. The method according to claim 28 or 29, wherein the step b) comprises determining if the test drug and/or agent is capable of decreasing and/or inhibiting the expression of the reporter gene.

31. The method according to any one of claims 28 to 30, wherein the p53 isoform gene is Δ113p53 gene.

32. The method according to any one of claims 28 to 31 , wherein the transgenic non-human animal is a transgenic fish.

33. The method according to any one of claims 28 to 32, wherein the transgenic non-human animal is a transgenic zebrafish.

34. The method according to any one of claims 28 to 33, wherein the reporter gene is selected from the group consisting of luciferase, galactosidase, chloramphenicol, acetyltransferase, b-glucuronidase, and alkaline phosphatase.

35. The method according to any one of claims 28 to 34, wherein the reporter gene is a fluorescent protein gene.

36. The method according to claim35, wherein the fluorescent protein is selected from the group consisting of GFP, RFP, BFP, YFP, and dsRED2.

37. The method according to claim 35, wherein the fluorescent protein is GFP.

38. The method according to any one of claims 28 to37, wherein the test drug and/or agent is a nucleic acid molecule capable of binding and/or hybridising to the p53 isoform gene, mRNA, promoter and/or a portion thereof.

39. The method according to any one of claims 28 to 38, wherein the test drug and/or agent is a nucleic acid molecule comprising a nucleotide complementary to and/or which hybridises to the p53 isoform gene, mRNA, promoter and/or a portion thereof.

40. The method according to any one of claims 28 to39, wherein the p53 isoform gene, mRNA, promoter and/or portion thereof is Δ113p53 gene, mRNA, promotor and/or a portion thereof.

41. The method according to any one of claims 38 to 40, wherein the nucleic acid molecule is an antisense nucleic acid molecule.

42. The method according to claims 41 , wherein the nucleic acid molecule is an antisense single strand RNA (sRNA), double strand RNA

(dsRNA), double strand DNA (dsDNA), double strand hybrid RNA/DNA (RNA/DNA), small interfering RNA (siRNA), micro RNA (miRNA), morpholinos (MO/PMO) and/or ribozymes.

43. The method according to claim 41 , wherein the nucleic acid molecule is morpholino (MO/PMO).

44. The method according to any one of claims 38 to43, wherein the nucleic acid molecule comprises a nucleotide sequence which is complementary to and/or which hybridizes to a 4113bp 5'-upstream region, or portion thereof, of the translation start site of the p53 isoform gene.

45. The according to any one of claims 38 to 43, wherein the nucleic acid molecule comprises a nucleotide sequence which is complementary to and/or which hybridizes to a 5'-upstream region -1041 to -1991bp or portion thereof of the transcription start site of the p53 isoform gene.

46. The according to any one of claims 38 to 43, wherein the nucleic acid molecule comprises a nucleotide sequence which is complementary to and/or which hybridizes to a 5'-upstream region -1 to -239bp or portion thereof of the transcription start site of the p53 isoform gene.