WO2001007478A1

WO2001007478A1 - A p1 artificial chromosome (pac) vector for the expression of pituitary adenyl cyclase activating peptide receptor (pacap receptor) and transgenic animals comprising said vector

Info

Publication number: WO2001007478A1
Application number: PCT/GB2000/001586
Authority: WO
Inventors: Sanbing Shen; Anthony John Harmar
Original assignee: Medical Research Council
Priority date: 1999-04-23
Filing date: 2000-04-25
Publication date: 2001-02-01
Also published as: GB9909446D0; AU4420200A

Abstract

A transgenic organism comprising a PAC vector is described. The PAC vector comprises a PACR gene, a reporter gene and an element capable of causing co-expression of the PACR gene and the reporter gene. The use of the PAC to study gene function and regulation in transgenic organisms is also described.

Description

A PI ARTIFICAL CHROMOSOME (PAC) VECTOR FOR THE EXPRESSION OF PITUITARY ADENYL CYCLASE ACTIVATING PEPTIDE RECEPTOR (PACAP RECEPTOR) AND TRANSGENIC ANIMALS COMPRISING SAID VECTOR

The present invention relates to a vector.

In particular, the present invention relates to a PI derived artificial chromosome (PAC) vector comprising pituitary adenylate cyclase activating polypeptide (PACAP) receptors.

More specifically, the invention relates to the use of a PAC vector comprising pituitary adenylate cyclase activating polypeptide (PACAP) receptors for the production of transgenic animals with widespread application in the study of PACAP receptor function and regulation.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide which is widely expressed in the brain, and in various peripheral organs, notably in endocrine glands, the gastro-intestinal and urogenital tracts and the respiratory system. In vivo and in vitro studies have shown that PACAP exerts multiple activities as a hormone, neurohormone, neurotransmitter, neuromodulator, vasodilator and neurotrophic factor. For instance, PACAP triggers the release of insulin and glucagon, activates steroidogenesis in the adrenal glands and gonads and stimulates the secretion of most hypophysial cells. PACAP exerts a potent relaxant activity on smooth muscle fibres in blood vessels, lung and gut. In the brain, PACAP stimulates the electrical activity of various populations of neurons and increases tyrosine hydroxylase gene expression. Recent studies have shown that PACAP exerts a trophic activity during ontogenesis, notably in the adrenal medulla and in the central nervous system (CNS). PACAP also a neurotrophic agent that may play an important role in the development of the brain. In the adult brain, PACAP appears to function as a neuroprotective factor that attenuates the neuronal damage resulting from various insults.

Post-translational processing of the PACAP precursor generates two biologically active molecular forms, PACAP38 and PACAP27 and a novel peptide called PACAP-related peptide whose activity remain unknown. The sequence of PACAP27 exhibits substantial similarities with those of vasoactive intestinal polypeptide (VIP), glucagon and secretin with the result that these neuropeptides are regarded as neuropeptides of the VIP/secretin/glucagon family.

The biological effects of PACAP are mediated through three distinct three G protein coupled receptors subtypes which exhibit differential affinities for PACAP and VIP. By way of example, one of these receptors (the PACl receptor or PACjR) is selective for PACAP while two of these receptors known as VPACi receptors and VPAC₂ receptors are also high-affinity receptors for VIP (Harmar et al, 1998). Synonyms for VPACi receptors include PACAP type 2, VlPi and PVR . Synonyms for VPAC₂ receptors include PACAP type 3, VIP₂ and PVR₃. It is recognised that PACiR is expressed at high level in brains but at a low level in the adrenals and scarcely in other principal tissues. In contrast, VPA receptor has been shown to be expressed in inter alia lungs, brains, small intestines, livers and VPAC₂ receptor has been shown to be expressed in inter alia lungs, stomachs, small intestines, and pancreas.

The complete sequence of the human PACiR gene with extensive regions of 5' and 3' flanking sequence has been published in GenBank (Accession no. AC006466: Waterston R.H.; "The sequence of Homo sapiens clone"; Genome Sequencing Center, Washington University School of Medicine, 4444 Forest Park Parkway, St. Louis, MO 63108, USA). AC006466 (in the version published in GenBank on 05-MAR-1999) consists of 184514 bp of DNA sequence consisting of 2 unordered pieces (1-78955 and 78973 -184514). The second of these pieces appears to contain the complete sequence of the human PAC R gene, with extensive regions of 5' and 3' flanking sequence. The location of the 18 exons of the human PACl receptor gene (17 coding exons and one exon 5' of the translated region called 5' UTR) within AC006466 is presented in Figure 1.

Multiple alternative transcripts of the gene can be produced by alternative splicing. By way of example, Pisegna and Wank (Pisegna and Wank 1996) have shown that there are six potential splice variants of the human PAC 1 R which differ (i) in the presence or absence of the 13th and 14th coding exons and (ii) in the use of one or other of two contiguous splice acceptor sites (a or b, Figure 2B) at the 5' end of exon 14, leading to the presence or absence of a serine residue at the N-teminus of the segment of the PACiR protein encoded by exon 14. The various receptor splice variants obtained were comparable in their ability to stimulate intracellular cyclic AMP accumulation but differed in their ability to stimulate intracellular inositol phosphate production, consistent with the fact that exons 13 and 14 encode amino acid sequences which lie within a region of the receptor (the third intracellular loop) that is thought to play an important role in the coupling of receptor to intracellular signalling pathways. Similar splice variation has been described in the rat (Spengler et al, 1993) and mouse (Aino et al, 1995).

Pantaloni et al (Pantaloni et al, 1996) have described splice variants of the mouse and human PACl receptors lacking exons 4 and 5, which encode a sequence of 21 amino acids within the N-terminal extracellular domain of the receptor. Removal of this 21 amino acid sequence modulated receptor selectivity with respect to PACAP-27 and PACAP-38 binding and influenced the relative potencies of the two agonists in phospholipase C stimulation.

Messenger RNA encoding the PACiR has been shown to be expressed predominantly in the CNS, most abundantly in the olfactory bulb, thalamus, hypothalamus, the dentate gyrus of the hippocampus and in granule cells of the cerebellum (Hashimoto et al., 1993; Spengler et al, 1993). In addition to acting as a neurotransmitter, there is an abundant literature suggesting that PACAP, acting through the PACiR, may function as a neurotrophic agent and neuroprotectant in vitro (Campard et al, 1997; Cavallaro et al, 1996; Gonzalez et al, 1997; Takei et al, 1998) and in vivo (Banks et al, 1996; Uchida et al, 1996).

In the periphery, the PACjR is present in the pituitary gland, pancreas, in the adrenal medulla (where PACAP is thought to function as a non-cholinergic neurotransmitter stimulating catecholamine secretion (Watanabe et al, 1995)) and ovary. In the mouse embryo, high concentrations of PACl R mRNA are expressed in the floor and roof plates of the neural tube, the rhombencephalon, the developing sympathetic chain and in the trigeminal ganglia, consistent with a role for PACAP or a related peptide in the early development of the central and peripheral nervous systems (Sheward et al, 1998; Sheward et al, 1996; Waschek et al, 1998). The PACiR is induced in mouse brain following transient focal cerebral ischemia, in parallel with a transcription factor (Zacl : Gillardon et al, 1998). Zac 1 is thought to transactivate the PAC i R gene, as well as regulating both apoptosis and cell cycle arrest. These observations indicate that agents that regulate the expression of the PACjR may be useful in the treatment of stroke and other cerebrovascular disorders.

Additional possible functions of the PACjR are suggested by the pharmacological properties of maxadilan, a potent agonist of the PACiR (Moro and Lerner, 1997). Maxadilan, a peptide from the salivary gland of the sand fly Lutzomyia longipalpis, a vector for leishmaniasis, is the most potent vasodilator known (Jackson et al, 1996), enhancing movement of the parasite into the body and blood into the parasite. Maxadilan also acts as an immune suppressant (Qureshi et al, 1996), facilitating infection by the parasite. These observations indicate that agents capable of regulating the expression or function of the human PACiR may be of value in the treatment or prevention of leishmaniasis, as immunosuppressants, or as vasodilators useful in the treatment of cardiovascular disease.

PACAP has been shown to function as a neuroprotectant in vitro in a number of regions of the CNS and periphery (that are prominent sites of PACiR expression) including cerebellar (Campard et al, 1997; Cavallaro et al, 1996; Chang et al, 1996; Gonzalez et al, 1997), cortical (Mono et al, 1996), hippocampal (Uchida et al, 1996), mesencephalic (Takei et al, 1998), sympathetic (Chang and Korolev, 1997) and sensory (Lioudyno et al, 1998) neurons. PACAP, administered either intracerebroventricularly or intravenously, has been reported to prevent ischaemia-induced death of hippocampal neurons in vivo (Banks et al, 1996; Uchida et al, 1996), even if the administration of PACAP was delayed for 24 h after the ischaemic event.

PACAP-immunoreactive nerve fibres have been described in the superficial layers of the dorsal horn (Dun et al, 1996; Moller et al, 1993; Narita et al, 1996; Zhang et al, 1997), presumably representing the nerve terminals of small sized, PACAP-containing neurons in the dorsal root ganglia (Moller et al, 1993; Zhang et al, 1997). The expression of PACAP in these neurons is up-regulated following nerve injury (Zhang et al, 1996). Capsaicin stimulates release of PACAP-like immunoreactivity from rat spinal cord in vivo (Zhang et al, 1997). Intrathecally administered PACAP has been reported to evoke behavior suggestive of hyperalgesia (Narita et al, 1996), but inhibited nociceptive behaviour in the rat formalin test which is considered to be a model of animal pain induced by inflammation (Yamamoto and Tatsuno, 1995).

PACAP has been shown to induce a long-lasting depression of transmission at the Schaffer collateral-CAl synapse while at the same time causing enhancement of the perforant path-granule cell synapse in the dentate gyrus (Kondo et al, 1997), suggesting that PACAP may play a role in learning and memory. The gene responsible for the Drosophila memory mutant amnesiac encodes a PACAP-like neuropeptide (Feany and Quinn, 1995), suggesting that such a role may have been widely conserved during evolution.

The in vivo functional analysis of genes such as PACiR frequently involve the introduction of native or modified genomic DNA into the germline to generate transgenic animals. The size of the genomic DNA that can be readily manipulated in vitro and introduced into the germline can be a critical determinant of the outcome of such experiments. Experiments from both Drosophila and mouse transgenesis have shown that elements that are important for high-level, tissue-specific and integration site independent expression of the transgene, such as enhancers, locus control regions (LCRs) and insulators may reside at a large distance (>50kb) from the gene itself.

Conventional transgenic studies using limited DNA fragments (<20kb) frequently results in low-level transgene expression and extensive position effects. Moreover, the large size of genes, such as the human PACiR gene (~50kb), means that standard transgenic technology using plasmid or cosmid DNA is inappropriate for transgenic animal studies. Thus, the ability to obtain large genomic DNA fragments, modify them and introduce the intact modified DNA into the germline is important for studying gene regulation and gene function in vivo.

One cloning system that allows easy manipulation of large genomic DNA is the yeast artificial chromosome (YAC). YACs contain on an average 500-600 kb of genomic DNA and have become important tools in physical mapping and in positional cloning of disease genes. However, there are several limitations to using YACs. Firstly, it is difficult to isolate intact YAC DNA. Secondly YACs have a high (40-50%) degree of chimerism and clonal instability. Thirdly, although several methods have been developed to generate YAC transgenics, purification of intact YAC DNA and preparation of intact YAC DNA and preparation of YAC transgenics carrying YAC DNA remain challenging tasks. WO 99/28449 (Application No. GB 98/03558) outlines ways in which some of these problems have been overcome.

As an alternative to YACs, E.coli based cloning systems have been developed to construct large genomic DNA insert libraries. By way of example, bacterial artificial chromosomes (BACs) are based on the E.coli fertility plasmid (F factor) and PI derived artificial chromosomes (PACs) are based on the bacteriophage PI (Ioannou et al 1994). BACs and PACs may propagate up to 300kb genomic DNA as 1 to 2 copy plasmids in a well characterised recombination deficient E.coli host strain. YACs, BACs, PACs, and MACs (mammalian artificial chromosomes) and their use as research tools are reviewed in Monaco et al (1994).

Despite the fact that BACs and PACs have many important applications (Nielsen et al 1999; Rouy et al 1998; Nielsen et al 1997; Smith et al 1995), with current expression monitoring techniques such as in situ hydridisation, Polymerase Chain Reaction (PCR) and Northern Blotting, it is not currently possible to readily monitor the in vivo expression pattern of a nucleotide sequence of interest (NOI) such as a PAC 1 R gene that has been introduced into an organism - such as a mouse. Moreover, current techniques - such as in situ hydridisation, Polymerase Chain Reaction (PCR) and Northern Blotting - are also laborious to carry out.

The present invention seeks to improve upon the existing techniques associated with the preparation of and usage of PACs to study gene function such as PACR gene function and regulation in transgenic mammals. The present invention also seeks to provide PACiR isoforms capable of expression under in-vitro and in-vivo conditions and which facilitate the study of PA R function and regulation in transgenic animals. SUMMARY ASPECTS

The present invention is the first report of the use of a modified PAC vector in the study of gene function and regulation in transgenic animals.

It is to be noted that none of the earlier reported studies has reported on the co-expression of NOIs such as a PAC R gene and a reporter gene in a PAC transgenic mammal.

In one aspect, the present invention provides transgenic mice that express a construct encoding the human PAC i R gene which enable studies on the regulation of the human PACiR gene expression in transgenic animal to be carried out. These studies facilitate the understanding of the functions of the PACiR gene in the developing and adult nervous system.

In another aspect, the present invention provides assay methods for studies on the regulation of PACl R gene expression in hPACiR-LαcZ mice in vivo and in vitro. A simple assay using LacZ reporter gene expression may be adaptable to a high throughput screening assay to facilitate the identification of agents capable of acting as PA R agonists and antagonists when administered to human subjects. Moreover, breeding of hPAClR-ZαcZ mice with PACjR knockout mice, can provide humanised" animals expressing the PACiR with human pharmacology, permitting the evaluation of the effectiveness of PAC R agonists and antagonists when administered to human subjects.

In another aspect, the present invention provides further sequence data extending 143bp upstream of the translation initiation (ATG) codon of the human PAC 1 receptor. The region upstream of the transcription start site of the human PACl receptor (SEQ ID No 3) contains DNA sequences that are the binding sites for transcription factors that regulate the expression of the PAC}R gene. Further analysis of this region allows the identification of such factors and permits the identification of treatments capable of regulating gene expression in vivo. Such strategies for the treatment of human disease can be easily tested in PACl receptor-IαcZ transgenic mice.

In yet another aspect, the present invention also demonstrates that up to 32 distinct splice variants of the PACl receptor may exist. In this respect:

(i) isoforms 25 - 32 have not been described previously and are of particular interest since they have a non-functional exon 3. These isoforms lack exon 3 which may encode a region of the gene which is thought to be necessary for ligand-activated signal transduction. This group of isoforms includes a splice variant of the PACjR gene called isoform 30 which was designated in UK patent application number 9909446.8 as isoform 18).

(ii) isoforms 9 - 16, comprising exon 3 and exon 5 and a non-functional exon 4 have not been described previously. These isoforms may have novel pharmacological properties since they lack exon 4 which is in a region of the PACiR which has been shown to influence the relative affinity of the receptor for PACAP-38, PACAP-27 and VIP.

(iii) isoforms 7, 15, 23 and 31 have non-functional exons 13, 14 and 15. These isoforms lack exons 13, 14 and 15. Isoforms 8, 16, 24 and 32 have non-functional exons 13, 14, 15 and 16. These isoforms lack these exons. These isoforms which have not been described previously are also of particular interest since they encode truncated receptors with 5 transmembrane domains rather than the 7 transmembrane domains of the full- length receptor. These isoforms may be impaired in their ability to respond to PACAP by coupling to intracellular signalling pathways.

In another aspect, the present invention demonstrates that the PA R is present in the dorsal horn of the spinal cord. Direct evidence for expression of the PA R in the dorsal horn of the spinal cord has recently been reported by Dickinson et al (1999). Earlier studies provided evidence for a role for PACAP but did not identify the receptor type involved. These data suggest that PACAP released from C-fibres in the spinal cord may play a role in modulating nociception. DETAILED ASPECTS OF THE INVENTION

In one aspect, the present invention relates to a transgenic organism comprising a PAC vector.

In a further aspect, the present invention relates to a transgenic organism comprising a PACR gene.

In a further aspect, the present invention relates to an isolated isoform of the human PACiR gene wherein the isolated isoform is selected from the group consisting of: isoforms 25-32; isoforms 9-16; isoforms 7, 15, 23 and 3; isoforms 8, 16, 24, and 32; or combinations thereof, preferably isoform 30.

In a further aspect, the present invention relates to a method for preparing a transgenic organism comprising providing (such as preparing) a PAC vector and inserting said vector into a transgenic organism thereby to form said transgenic organism.

In a further aspect, the present invention relates to the use of a PAC and/or a PACiR gene to study gene function and regulation in a transgenic organism.

In a further aspect, the present invention relates to the use of a PAC and/or a PA R gene to facilitate the understanding of the functions of the PACiR gene in the developing nervous system and the adult nervous system.

In a further aspect, the present invention relates to the use of a PAC and/or a PACiR gene to screen for agents capable of affecting PA R activity or the expression pattern of the PACiR gene in a transgenic organism.

In a further aspect, the present invention relates to the use of a PAC and/or a PACiR gene to screen for agents capable of affecting the expression pattern of the human PA R gene in a transgenic organism wherein the expression pattern of the human PACjR gene in the central nervous system (CNS) is in a tissue-specific and developmentally regulated pattern that closely mimics that of the endogenous mouse PA R gene. In a further aspect, the present invention relates to the use of a transgenic organism comprising a PAC and or a PACiR gene to test for potential pharmaceutical and/or veterinary agents.

In a further aspect, the present invention relates to the use of a PAC vector and/or a PACiR gene and/or expression products thereof in the preparation of a medicament in the treatment and/or modulation of disturbances in any one of: stroke and other cerebovascular disorders; cardiovascular disease; leishmaniasis; immunosuppressive disorders; nociception; and learning and memory functions.

In a further aspect, the present invention relates to a PAC mouse.

These aspects an further aspects of the invention are presented in the accompanying claims and in the following description and drawings. For ease of reference, these aspects and further aspects are presented under separate section headings. However, it is to be understood that the teachings under each section are not necessarily limited to that particular section heading. These aspects and other aspects are now discussed.

SURPRISING AND UNEXPECTED FINDINGS

The present invention demonstrates the surprising and unexpected findings that:

(i) in transgenic mice comprising the PAC construct of the present invention, LacZ was expressed in a tissue-specific and developmentally regulated pattern that closely mimics that of the endogenous mouse PACiR. Consistent with previous studies using in situ hybridization (Hashimoto et al, 1996; Shioda et al, 1997), the most prominent site of expression of the LacZ reporter gene in human PACjR-ZαcZ mice was the hippocampus, most intensely in the dentate gyrus.

(ii) the PAC R is present in the dorsal horn of the spinal cord. These data suggest that PACAP released from C-fibres in the spinal cord may play a role in modulating nociception. (iii) up to 32 distinct splice variants of the PACl receptor may exist some of which have not been previously described.

(iv) PAC i receptor isoforms with non-functional exon 3 or exon 15 may be unresponsive to PACAP. In particular, isoform 30 - designated in UK patent application number 9909446.8 as isoform 18) which has not been described previously, lacks a region of the gene (exon 3) which is thought to be necessary for ligand-activated signal transduction.

(v) PACl receptor isoforms with a non- functional exon 4 may differ from other isoforms in their responsiveness to PACAP or other ligands.

(vi) transgenic mice expressing the PA receptor at very high levels may display a developmental defect.

ADVANTAGES

The present invention is advantageous because:

(i) it is possible to readily monitor the expression pattern of an NOI such as a PACiR gene.

(ii) it provides a means for producing PAC transgenic mammals and the analysis of these mammals in terms of expression, regulation and function of an NOI such as a PACiR gene.

(iii) it facilitates the determination of sites/regions where an NOI such as a PACiR gene is expressed and the identification of agents which may affect the expression pattern of an NOI such as a PACiR gene and or the activity of the expression product (PACiR) thereof.

(iv) it demonstrates that the introduction into the germ line of transgenic mice of a PAC vector encoding the promoter and coding region of the human PACiR gene linked to a reporter gene such as LacZ, results in a position-independent expression of Z, cZ in the CNS in a pattern that closely resembles that of the endogenous mouse PAC R gene. (v) it demonstrates that the transgenic approach provides a more complete picture of the pattern of expression of the PACjR gene than standard procedures such as in situ hybridisation.

(vi) it demonstrates that the transgenic approach facilitate studies on the developmental and physiological regulation of PACjR gene expression in vitro and in vivo.

(vii) it demonstrates that agents that regulate the expression of the PACiR can be easily identified. These agents can form the basis of new treatments for PACiR disorders relating to learning and memory, ischemic brain injury and nociception (reaction to pain sensation).

Other advantages associated with the present invention are made apparent in the commentary.

PI -DERIVED ARTIFICIAL CHROMOSOME (PAC)

As used herein, the term "PAC" means a PI -derived artificial chromosome. The PAC is a cloning system for isolating genomic DNA based on the F-factor plasmid, as in BACs. The PAC may contain one or more elements of the bacteriophage PI cloning system. With the present invention, the PAC or PAC vector has a modified genetic structure. In a preferred aspect, an IRES and a reporter gene has been incorporated into the PAC or PAC vector. Here the term "modified PAC or modified PAC vector" means a modified PAC or modified PAC vector having a modified genetic structure.

BACTERIAL ARTIFICIAL CHROMOSOME (BAC)

As used herein, the term "BAC" means a bacterial artificial chromosome which is a cloning system for isolating genomic DNA based on the F-factor plasmid. The F-factor plasmid is the bacterial sex or fertility plasmid which has a low copy number because of the strict control of replication. PAC VECTOR

With the present invention, the PAC vector may comprise at least one PACiR gene.

The PAC vector of the present invention can be used, for example, for expression and/or regulation and/or functional studies of a PACR gene such as a PAC]R gene. In addition, the PAC vector of the present invention can be used to prepare transgenic organisms that can be used for expression and/or regulation and/or functional studies of the PACiR gene in combination with other entities, such as other NOIs, compounds or compositions. In addition, the PAC or PAC vector of the present invention can be used to test potential pharmaceutical agents (including veterinary agents).

The PAC vector of the present invention may be used to prepare transformed cells that can be used, for example, for functional studies of the PACiR gene. In addition, the PAC vector of the present invention can be used to prepare transformed cells that can be used for functional studies of the PA R gene in combination with other entities, such as other NOIs, compounds or compositions. In addition, the transformed cells can be used to test potential pharmaceutical agents (including veterinary agents).

PAC RECEPTOR (PACR)

In the context of the present invention, the term "PACR" of the present invention refers to PACAP type 1 receptors (PA R), VPACi receptors and VPAC₂ receptors.

PACAP TYPE 1 RECEPTOR (PAC,R)

The term "PACjR" of the present invention refers to PACAP type 1 receptors which are membrane-bound proteins existing in the nervous system - particularly brain hypothalami and pituitary glands of various mammalian species - and in various peripheral organs, such as the endocrine glands, the adrenals, the gastrointestinal and urogenital tracts and the respiratory system. The term includes isoforms/splice variants thereof. In accordance with the present invention, the "PA R" can be the protein er se also as well as being part of a fusion protein.

PACiR ALLELES

Included within the scope of the present invention are alleles of the PAdR. As used herein, an "allele" or "allelic sequence" is an alternative form of the PA R. Alleles result from a mutation, i.e., a change in the nucleotide sequence, and generally produce altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given gene may have none, one or many allelic forms. Common mutational changes which give rise to alleles are generally ascribed to deletions, additions or substitutions of amino acids. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.

ISOFORM

The term "isoform" means any of several multiple forms of the same protein that differ in their primary structure but which may not retain the same function. They can be produced by alternative splicing of RNA transcripts from the same gene. In the context of the present invention, the term "isoform" is used interchangeably with the term "splice variant". The present invention demonstrates that up to 32 isoforms of the PACi receptor have been identified. Some of these isoforms have not been previously described. Some of these PACi receptor isoforms do not retain the same function as the PA receptor. By was of example, PACi receptor isoforms that have at least a non-functional exon 3 or a non-functional exon 15 may be unresponsive to PACAP. In this respect, isoform 30 (previously designated in UK patent application number 9909446.8 as isoform 18) has a non-functional exon 3 because it lacks an exon 3.

EXON

The term "exon" means any intragenic region of a DNA sequence that will be ultimately expressed in (mature) mRNA or rRNA residues. The location of the 18 exons of the human PACj receptor gene (17 coding exons and one exon 5' of the translated region called 5' UTR) within AC006466 is presented in Figure 1. In the context of the present invention, the terms exon 1, exon 2, exon 3 etc are used in accordance with the exon classification system as set out in Figure 1.

NON-FUNCTIONAL EXON

As used herein, with reference to the term "exon", the term "non-functional" includes but is not limited to an exon which may be removed, mutated, silenced and/or inactivated so that it is no longer expressed. The term also includes but is not limited to an exon which may have an altered open reading frame (ORF) so that it is expressed in a truncated form.

NUCLEOTIDE SEQUENCES

The present invention also relates to DNA fragments of the sequences of the present invention for use as probes.

The present invention also relates to DNA fragments comprising the DNA sequence of SEQ ID No. 1 or allelic variations of such sequences.

A preferred - but non-limiting - example of a PACi R gene is the human PACjR

(hPACiR) gene presented as SEQ ID No. 1 or a variant, homologue or derivative thereof.

A preferred- but non limiting example- of a PA R is the protein comprising the amino acid sequence of SEQ ID No. 2.

The terms "variant", "homologue", or "derivative" in relation to this aspect of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant expression product of the nucleotide sequence has the same activity as the expression product of SEQ ID No. 1, preferably having at least the same level of activity of the expression product of SEQ I.D. No 1. In particular, the term "homologue" covers identity with respect to structure and/or function providing the the resultant expression product of the nucleotide sequence has the same activity as the expression product of SEQ ID No. 1, preferably having at least the same level of activity of the expression product of SEQ I.D. No 1. With respect to sequence identity (i.e. similarity), preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% sequence identity. More preferably there is at least 95%, more preferably at least 98%, sequence identity. These terms also encompass allelic variations of the sequences.

A highly preferred aspect of the present invention relates to isoform 30 (previously designated in UK patent application number 9909446.8 as isoform 18) of human PACiR comprising the amino acid sequence of SEQ ID No. 6. For example, the present invention relates to an isolated human PACjR comprising the amino acid sequence of SEQ ID No. 6 or a variant, homologue or derivative thereof.

The present invention also relates to DNA comprising the DNA sequence of SEQ ID No. 7 or a variant, homologue or derivative thereof.

The terms "variant", "homologue", or "derivative" in relation to this aspect of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant expression product of the nucleotide sequence has the same activity as the expression product of SEQ ID No. 7, preferably having at least the same level of activity of the expression product of SEQ I.D. No 7. In particular, the term "homologue" covers identity with respect to structure and/or function providing the the resultant expression product of the nucleotide sequence has the same activity as the expression product of SEQ ID No. 7, preferably having at least the same level of activity of the expression product of SEQ I.D. No 7. With respect to sequence identity (i.e. similarity), preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% sequence identity. More preferably there is at least 95%, more preferably at least 98%, sequence identity. These terms also encompass allelic variations of the sequences.

The present invention also relates to non-native DNA comprising the DNA sequence of SEQ ID No. 7 or an allelic variation thereof. ISOLATED PAC,R ISOFORMS

In one preferred embodiment, the nucleotide sequence and/or the PA R (isoform 30 - previously designated in UK patent application number 9909446.8 as isoform 18) thereof are in an isolated and/or purified form.

In one preferred embodiment, the novel isoforms of the human PACiR of the present invention, selected from the group consisting of: isoforms 25-32; isoforms 9-16; isoforms 7, 15, 23 and 3; isoforms 8, 16, 24, and 32; or combinations thereof are in an isolated and/or purified form.

As used herein, the terms "isolated" and "purified" refer to molecules, either nucleic or amino acid sequences, that are removed from their natural environment and/or isolated and/or separated from at least one other component with which they are naturally associated.

The isolated isoform 30 - designated in UK patent application number 9909446.8 as isoform 18) of the human PACiR of the present invention can be produced by recombinant DNA methods or synthetic peptide chemical methods that are well known to those of ordinary skill in the art. Protein purification methods are also well known in the art.

The isolated isoforms selected from the group consisting of: isoforms 25-32; isoforms 9- 16; isoforms 7, 15, 23 and 3; isoforms 8, 16, 24, and 32 of the human PACiR of the present invention can be produced by recombinant DNA methods or synthetic ^"peptide chemical methods that are well known to those of ordinary skill in the art. Protein purification methods are also well known in the art.

EXPRESSION IN A HOST CELL

The present invention also relates to isoform 30 - previously designated in UK patent application number 9909446.8 as isoform 18) of the human PACiR produced by expression in a transformed host cell into which has been incoφorated the foregoing DNA sequences or allelic variations thereof.

The present invention also relates to isoforms selected from the group consisting of: isoforms 25-32; isoforms 9-16; isoforms 7, 15, 23 and 3; isoforms 8, 16, 24, and 32 of the human PACiR of the present invention produced by expression in a transformed host cell into which has been incorporated the foregoing DNA sequences or allelic variations thereof.

VECTORS

The term "vector" includes expression vectors and transformation vectors.

The term "expression vector" means a construct capable of in vivo or in vitro expression.

The term "transformation vector" means a construct capable of being transferred from one species to another.

As it is well known in the art, a vector is a tool that allows or faciliates the transfer of an entity from one environment to another. In accordance with the present invention, and by way of example, some vectors used in recombinant DNA techniques allow entities, such as a segment of DNA (such as a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a target cell. Optionally, once within the target cell, the vector may then serve to maintain the heterologous DNA within the cell or may act as a unit of DNA replication. Examples of vectors used in recombinant DNA techniques include plasmids, chromosomes, artificial chromosomes or viruses. The artificial chromosomes include but are not limited to PACs, BACs, YACs and MACs.

The present invention also encompasses PAC vectors comprising these aspects of the present invention, transformed cells comprising these aspects of the present invention, transgenic organisms comprising these aspects of the present invention, processes for making all of these aspects, and methods of expressing all of these aspects. AGENT

The term "agent" includes any entity (such as one or more chemical compounds, including peptide sequences and variants/homologues/derivatives/fragments thereof) which is capable of affecting the expression pattern of the PACiR gene or the activity of the PACjR expression product thereof. It also includes mimics and equivalents and mutants thereof. It also includes agonists and antagonists and antibodies. Non-limiting antibodies include: polyclonal, monoclonal, chimeric, single chain, Fab fragments, fragments produced by a Fab expression library and humanised monoclonal antibodies.

AFFECTS

The term "affects" includes any one or more of: treats, prevents, suppresses, alleviates, restores, modulates, influences or to otherwise alter an existing state.

EXPRESSION PRODUCT (EP)

The term "expression product" or "EP" means the expressed protein per se but also includes fusion proteins comprising all or part of same. The EP may be the same as the naturally occuring form or is a variant, homologue, fragment or derivative thereof.

TRANSFORMED CELLS

The vectors of the present invention may be used to prepare transformed cells that comprise mutated genes - such as by use of the pop-in/pop-out technique.

Here the term "transformed cell" means a cell having a modified genetic structure.

With the present invention, the cell has a modified genetic structure since a vector according to the present invention has been introduced into the cell.

The term "cell" includes a cell from any suitable organism. In a preferred embodiment, the cell is a mammalian cell. In a highly preferred embodiment, the cell is a murine cell. The cell can be an isolated cell or a collection of cells. The cell or cells may even be part of a tissue or organ or an organism (including an animal).

The cell can be transformed in vivo, ex-vivo or in vitro, or combinations thereof.

TRANSFORMATION METHODS

Typically, the cell will be transformed by any one of the following methods which include but are not limited to: transfection, transduction, microinjection, or microprojectile bombardment, including combinations thereof.

TRANSFECTION METHODS

Preferably, the cell will be transformed by, or by at least, transfection.

Typical transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent-mediated, cationic facial amphiphiles (CFAs) (Nature Biotechnology 1996 14; 556), multivalent cations such as spermine, cationic lipids or polylysine, 1, 2,-bis (oleoyloxy)-3-(trimethylammonio) propane (DOTAP)-cholesterol complexes (Wolff and Trubetskoy 1998 Nature Biotechnology 16: 421) and combinations thereof.

VIRAL DELIVERY SYSTEMS

The cells may also be tranduced by viral delivery systems.

Viral delivery systems include but are not limited to adenovirus vector, an adeno- associated viral (AAV) vector, a herpes viral vector, a retroviral vector, a lentiviral vector or a baculoviral vector. TRANSGENIC ORGANISMS

For some applications, the transformed cells may be prepared by use of the PAC vector according to the present invention. 5

The PAC vector of the present invention can also be used to prepare transgenic organisms that can be used for functional studies of the PACiR. In addition, the vectors of the present invention can be used to prepare transgenic organisms that can be used, for example, for functional studies of the PACiR in combination with other entities, such as 10 expression products of other NOIs, compounds or compositions. In addition, the transgenic organisms can be used to test potential pharmaceutical agents (including veterinary agents).

Here the term "transgenic organism" means an organism comprising a modified genetic 15 structure. With the present invention, the organism has a modified genetic structure since a vector according to the present invention has been introduced into the organism.

The term "organism" includes any suitable organism. In a preferred embodiment, the organism is a mammal. The term "mammal" includes but is not limited to humans, 20 primates, rats, mice, guinea pigs, rabbits, horses, cows, sheep, pigs, goats and the like.

In a highly preferred embodiment, the organism is a mouse.

For some applications, the transgenic organisms may be prepared by use of the 25 transformed cells of the present invention.

SELECTION GENES

With the present invention, the PAC vector of the present invention may additionally 30. comprise one or more selection genes to enable the vector and any resultant entity comprising the same or made from the same vector to be selectively grown and/or screened. These selection genes can be chosen from suitable selection genes that are available. In a preferred aspect any one or more of the selection gene is specifically removable from the PAC vector according to the present invention. In this regard, the term "specifically removable" means being able to remove the one or more selection gene without disrupting any other region in the PAC vector according to the present invention. For example, the selection gene may be flanked by unique restriction sites. Alternatively, the selection gene may be flanked by a LoxP element which is removable by use of Cre recombinase. Teachings on the use of the LoxP element and Cre recombinase have been published by Deursen et al (1995 PNAS Vol 93 pages 7376-7380), Kuhn et al (1995 Science Vol 269 pages 1427-1429) and Araki et al (1995 PNAS Vol 92 pages 160-164). By way of example, the selection gene flanked by the LoxP element may therefore be removed prior to or after formation of the transgenic animal stem cell. Removal of the selection gene is highly desirable as it means that the transgenic organism is not expressing the selection gene and so there can be no effect of that gene on the organism or even on the expression of the NOI being studied. In addition, removal of the selection gene means that the NOI is nearer to any 3' regulatory regions that may be present on the PAC.

NUCLEOTIDE SEQUENCES OF INTEREST (NOIs)

With the present invention, the PAC vector may additionally comprise one or more NOIs. The NOI need not necessarily be a complete naturally occuring DNA sequence. Thus, the DNA sequence can be, for example, a synthetic DNA sequence, a recombinant DNA sequence (i.e. prepared by use of recombinant DNA techniques), a cDNA sequence or a partial genomic DNA sequence, including combinations thereof. The DNA sequence need not be a coding region. If it is a coding region, it need not be an entire coding region. In addition, the DNA sequence can be in a sense orientation or in an anti-sense orientation. Preferably, it is in a sense orientation. Preferably, the DNA is or comprises cDNA. The NOI need not be of known function and/or structure. By way of example, the PAC or PAC vector may comprise at least two NOIs. Of these these NOIs, may be a NOI of human origin such as a human PA R gene or isoforms/splice variants thereof. The other of these NOI may be reporter gene such as a LacZ reporter gene. Alternatively, the NOI may comprise a promoter/enhancer sequence with a regulatory role. Preferably, the NOI is of human origin.

The present invention also relates to DNA fragments comprising the DNA sequence presented as SEQ ID No. 3 or allelic variations of such sequences.

A preferred - but non-limiting - example of a sequence comprising binding sites for factors capable of regulating expression of a human PACjR gene or isoforms/splice variants thereof is presented as SEQ ID No. 3 or a variant, homologue or derivative thereof.

The terms "variant", "homologue", or "derivative" in relation to this aspect of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to sequence providing the resultant nucleotide sequence has regulatory activity, preferably having at least the same regulatory activity as SEQ ID No. 3. In particular, the term "homologue" covers identity with respect to structure and/or function providing the the resultant nucleotide sequence has the same regulatory activity as SEQ ID No. 3, preferably having at least the same level of regulatory activity of the expression product of SEQ I.D. No 3. With respect to sequence identity (i.e. similarity), preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% sequence identity. More preferably there is at least 95%, more preferably at least 98%, sequence identity. These terms also encompass allelic variations of the sequences.

INTERNAL RIBOSOMAL ENTRY SITE (IRES).

In accordance with the present invention, the PAC vector comprises an internal ribosomal entry site (i.e. an IRES).

A review on IRES is presented by Mountford and Smith (TIG May 1995 vol 11 No. 5 pages 179 - 184). A suitable IRES has also been disclosed by Mountford et al (Mountford et al 1994 PNAS 91 pages 4303-4307). IRES sequences are also mentioned in WO-A-93/03143, WO-A-97/14809, WO-A- 94/24301, WO-A-95/32298, and WO-A-96/27676. These references do not disclose or suggest the use of an IRES unit in preparing or being a part of a PAC.

Yang et al (1997) discloses a BAC modification system using a homologous recombination method to insert an IRES-IαcZ marker gene. Yang et al does not report on the co-expression of NOIs, such as the PACiR gene and a reporter gene in a PAC transgenic organism.

According to WO-A-97/14809, IRES sequences act on improving translation efficiency of RNAs in contrast to a promoter's effect on the transcription of DNAs. A number of different IRES sequences are known including those from encephalomyocarditis virus (EMCV) (Ghattas, I.R., et al, Mol Cell. Biol., 11 :5848-5859 (1991); BiP protein [Macejak and Sarnow, Nature 353:91 (1991)]; the Antennapedia gene of drosphilia (exons d and e) [Oh, et al, Genes & Development, 6:1643-1653 (1992)] as well as those in polio virus [Pelletier and Sonenberg, Nature 334: 320-325 (1988); see also Mountford and Smith, TIG 11, 179-184 (1985)].

According to WO-A-97/14809, IRES sequences are typically found in the 5' non-coding region of genes. In addition to those in the literature they can be found empirically by looking for genetic sequences that affect expression and then determining whether that sequence affects the DNA (i.e. acts as a promoter or enhancer) or only the RNA (acts as an IRES sequence).

Thus the present invention is not intended to be limited to a specific IRES sequence. Instead, the sequence to be used can be any sequence that is capable of acting as an IRES sequence - i.e. it is capable of improving translation efficiency of an RNA.

A preferred IRES sequence is that presented as SEQ ID No. 4 or a variant, homologue, derivative or fragment thereof.

The terms "variant", "homologue", "derivative" or "fragment" in relation to this aspect of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence has IRES activity, preferably having at least the same activity of the IRES shown as SEQ I.D. No. 4. In particular, the term "homologue" covers identity with respect to structure and/or function providing the resultant nucleotide sequence has IRES activity. With respect to sequence identity (i.e. similarity), preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% sequence identity. More preferably there is at least 95%, more preferably at least 98%, sequence identity. These terms also encompass allelic variations of the sequences.

Another preferred IRES sequence is that presented as SEQ ID No. 5 or a variant, homologue, derivative or fragment thereof.

The terms "variant", "homologue", "derivative" or "fragment" in relation to this aspect of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence has IRES activity, preferably having at least the same activity of the IRES shown as SEQ I.D. No. 5. In particular, the term "homologue" covers identity with respect to structure and/or function providing the resultant nucleotide sequence has IRES activity. With respect to sequence identity (i.e. similarity), preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% sequence identity. More preferably there is at least 95%, more preferably at least 98%, sequence identity. These terms also encompass allelic variations of the sequences.

Sequence identity with respect to any of SEQ ID 1-7 can be determined by a simple "eyeball" comparison (i.e. a strict comparison) of any one or more of the sequences with another sequence to see if that other sequence has, for example, at least 75% sequence identity to the sequence(s).

Relative sequence identity can also be determined by commercially available computer programs that can calculate % identity between two or more sequences using any suitable algorithm for determining identity, using for example default parameters. A typical example of such a computer program is CLUSTAL. Advantageously, the BLAST algorithm is employed, with parameters set to default values. The BLAST algorithm is described in detail at http://www.ncbi.nih.gov/BLAST blast_help.html, which is incoφorated herein by reference. The search parameters are defined as follows, can be advantageously set to the defined default parameters.

5 Advantageously, "substantial identity" when assessed by BLAST equates to sequences which match with an EXPECT value of at least about 7, preferably at least about 9 and most preferably 10 or more. The default threshold for EXPECT in BLAST searching is usually 10.

10 BLAST (Basic Local Alignment Search Tool) is the heuristic search algorithm employed by the programs blastp, blastn, blastx, tblastn, and tblastx; these programs ascribe significance to their findings using the statistical methods of Karlin and Altschul (see http://www.ncbi.nih.gov/BLAST/blast_help.html) with a few enhancements. The BLAST programs were tailored for sequence similarity searching, for example to identify

15 homologues to a query sequence. For a discussion of basic issues in similarity searching of sequence databases, see Altschul et al (1994) Nature Genetics 6:119-129.

The five BLAST programs available at http://www.ncbi.nlm.nih.gov perform the following tasks: 20 blastp - compares an amino acid query sequence against a protein sequence database.

blastn - compares a nucleotide query sequence against a nucleotide sequence database.

25 blastx - compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database.

tblastn - compares a protein query sequence against a nucleotide sequence database dynamically translated in all six reading frames (both strands).

J 0 tblastx - compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database. BLAST uses the following search parameters:

HISTOGRAM - Display a histogram of scores for each search; default is yes. (See parameter H in the BLAST Manual).

DESCRIPTIONS - Restricts the number of short descriptions of matching sequences reported to the number specified; default limit is 100 descriptions. (See parameter V in the manual page).

EXPECT - The statistical significance threshold for reporting matches against database sequences; the default value is 10, such that 10 matches are expected to be found merely by chance, according to the stochastic model of Karlin and Altschul (1990). If the statistical significance ascribed to a match is greater than the EXPECT threshold, the match will not be reported. Lower EXPECT thresholds are more stringent, leading to fewer chance matches being reported. Fractional values are acceptable. (See parameter E in the BLAST Manual).

CUTOFF - Cutoff score for reporting high-scoring segment pairs. The default value is calculated from the EXPECT value (see above). HSPs are reported for a database sequence only if the statistical significance ascribed to them is at least as high as would be ascribed to a lone HSP having a score equal to the CUTOFF value. Higher CUTOFF values are more stringent, leading to fewer chance matches being reported. (See parameter S in the BLAST Manual). Typically, significance thresholds can be more intuitively managed using EXPECT.

ALIGNMENTS - Restricts database sequences to the number specified for which high- scoring segment pairs (HSPs) are reported; the default limit is 50. If more database sequences than this happen to satisfy the statistical significance threshold for reporting (see EXPECT and CUTOFF below), only the matches ascribed the greatest statistical significance are reported. (See parameter B in the BLAST Manual).

MATRIX - Specify an alternate scoring matrix for BLASTP, BLASTX, TBLASTN and TBLASTX. The default matrix is BLOSUM62 (Henikoff & Henikoff, 1992). The valid alternative choices include: PAM40, PAM120, PAM250 and IDENTITY. No alternate scoring matrices are available for BLASTN; specifying the MATRIX directive in BLASTN requests returns an error response.

STRAND - Restrict a TBLASTN search to just the top or bottom strand of the database sequences; or restrict a BLASTN, BLASTX or TBLASTX search to just reading frames on the top or bottom strand of the query sequence.

FILTER - Mask off segments of the query sequence that have low compositional complexity, as determined by the SEG program of Wootton & Federhen (1993) Computers and Chemistry 17:149-163, or segments consisting of short-periodicity internal repeats, as determined by the XNU program of Claverie & States (1993) Computers and Chemistry 17:191-201, or, for BLASTN, by the DUST program of Tatusov and Lipman (see http://www.ncbi.nlm.nih.gov). Filtering can eliminate statistically significant but biologically uninteresting reports from the blast output (e.g., hits against common acidic-, basic- or proline-rich regions), leaving the more biologically interesting regions of the query sequence available for specific matching against database sequences.

Low complexity sequence found by a filter program is substituted using the letter "N" in nucleotide sequence (e.g., "NNNNNNNNN NNN") and the letter "X" in protein sequences (e.g., "XXXXXXXXX").

Filtering is only applied to the query sequence (or its translation products), not to database sequences. Default filtering is DUST for BLASTN, SEG for other programs.

It is not unusual for nothing at all to be masked by SEG, XNU, or both, when applied to sequences in SWISS-PROT, so filtering should not be expected to always yield an effect. Furthermore, in some cases, sequences are masked in their entirety, indicating that the statistical significance of any matches reported against the unfiltered query sequence should be suspect. NCBI-gi - Causes NCBI gi identifiers to be shown in the output, in addition to the accession and/or locus name.

Most preferably, sequence comparisons are conducted using the simple BLAST search algorithm provided at http://www.ncbi.nlm.nih.gov/BLAST.

Other computer program methods to determine identify and similarity between the two sequences include but are not limited to the GCG program package (Devereux et al 1984 Nucleic Acids Research 12: 387) and FASTA (Atschul et al 1990 J Molec Biol 403-410).

In some aspects of the present invention, no gap penalties are used when determining sequence identity.

PROBES

The present invention also encompasses nucleotide sequences that are complementary to the sequences presented herein, or any fragment or derivative thereof. If the sequence is complementary to a fragment thereof then that sequence can be used as a probe to identify similar promoter sequences in other organisms etc.

HYBRIDISATION

The present invention also encompasses nucleotide sequences that are capable of hybridising to the sequences presented herein, or any fragment or derivative thereof.

Hybridization means a "process by which a strand of nucleic acid joins with a complementary strand through base pairing" (Coombs J (1994) Dictionary of Biotechnology, Stockton Press, New York NY) as well as the process of amplification as carried out in polymerase chain reaction technologies as described in Dieffenbach CW and GS Dveksler (1995, PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview NY). Also included within the scope of the present invention are nucleotide sequences that are capable of hybridizing to the nucleotide sequences presented herein under conditions of intermediate to maximal stringency. Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego CA), and confer a defined "stringency" as explained below.

Maximum stringency typically occurs at about Tm-5°C (5°C below the Tm of the probe); high stringency at about 5°C to 10°C below Tm; intermediate stringency at about 10°C to 20°C below Tm; and low stringency at about 20°C to 25 °C below Tm. As will be understood by those of skill in the art, a maximum stringency hybridization can be used to identify or detect identical nucleotide sequences while an intermediate (or low) stringency hybridization can be used to identify or detect similar or related nucleotide sequences.

In a preferred aspect, the present invention covers nucleotide sequences that can hybridise to the nucleotide sequences of the present invention under stringent conditions (e.g. 65°C and O.lxSSC).

The present invention also encompasses nucleotide sequences that are capable of hybridising to the sequences that are complementary to the sequences presented herein, or any fragment or derivative thereof. Likewise, the present invention encompasses nucleotide sequences that are complementary to sequences that are capable of hybridising to the sequence of the present invention. These types of nucleotide sequences are examples of variant nucleotide sequences. In this respect, the term "variant" encompasses sequences that are complementary to sequences that are capable of hydridising to the nucleotide sequences presented herein. Preferably, however, the term "variant" encompasses sequences that are complementary to sequences that are capable of hydridising under stringent conditions (eg. 65°C and O.lxSSC { lxSSC - 0.15 M NaCl, 0.015 Na₃ citrate pH 7.0}) to the nucleotide sequences presented herein.

Insertion of the IRES into a PAC by use of the insertion vector of the present invention - and thus forming a modified PAC - enables the modified PAC to express, in particular over-express, at least two nucleotide sequences. Of these two nucleotide sequences one 31 may be a NOI, such as a NOI of human origin such as a human PACiR gene or isoforms/splice variants thereof. The other of those nucleotide sequences may be another NOI. Alternatively, and in a preferred aspect, the other nucleotide sequence is a reporter gene according to the present invention.

The present invention also encompasses a PAC vector comprising more than one IRES. In this embodiment, the PAC vector preferably comprises more than NOI.

REPORTERS

In a preferred aspect of the first aspect of the present invention, the PAC vector comprises a reporter gene whose expression product is capable of producing a visually detectable signal. Examples of such reporter genes include: LacZ (see Mansour et al 1990 PNAS vol 87 pp 7688-7692), green fluorescent protein (see Chiocchetti et al 1997 Biochim Biophys Acta 1352: pp 193-202; and Chalfie et al 1994 Science vol 263 pp 802-805), chloroamphenicol acetyl transferase (see Gorman et al 1982 Mol Cell Biol 2(9) pp 1044- 1051; and Frebourg and Brison 1988 Gene vol 65 pp 315-318), or luciferase (see de Wet et al 1987 Mol Cell Biol 7(2) pp 725-737; and Rodriguez et al 1988 PNAS vol 85 pp 1667-1671).

In another preferred embodiment of the first aspect of the present invention, the PAC vector comprises a reporter gene whose expression product is capable of producing, or being detected by an agent capable of providing, an immunologically detectable signal.

In a preferred aspect, the reporter gene when fused to the NOI leads to the production of a fusion protein that can be detected by commercially available antibodies, such as a haemagglutinin tag (see Pati 1992 Gene 15; 114(2): 285-288), a c-myc tag (see Emrich et al 1993 Biocem Biophys Res Commun 197(1): 214-220), or the FLAG epitope (Ford et al 1991 Protein Expr Purif Apr; 2(2):95-107).

By using a reporter gene according to the present invention it is possible to readily observe the functionality of NOIs contained within PAC libraries, such as PAC human DNA libraries. 32 For example, if the NOI in the PAC has an expression regulatory role (such as a promoter) then expression of the reporter gene according to the present invention by a transgenic organism according to the present invention enables workers to readily determine in or at which sites or regions that expression regulatory element is active. In addition, workers will be able to readily test agents etc. that may affect the expression ability or pattern of that regulatory element.

By way of further example, if the NOI in the PAC has a functional role other than an expression regulatory role then workers can fuse (either directly or indirectly such as by means of one or more spacing nucleotide sequences) the NOI to the reporter gene according to the present invention. Thus, if the NOI is fused to the reporter gene according to the present invention and is present in a transgenic organism according to the present invention, then workers can readily determine which sites or regions that NOI is expressed. In addition, workers will be able to readily test agents etc. that may affect the expression pattern of that NOI.

A further advantage is that by being able to readily monitor the expression pattern or level of the NOI enables workers to determine the phenotype associated with the NOI.

EXAMPLES

The present invention will now be described only by way of example in which reference shall be made to the following Figures:

Figure 1 which shows the location of the 18 exons of the human PACjR receptor gene (17 coding exons and one exon 5' of the translated region called 5'UTR within the GenBank sequence published as Accession No. AC006466.

Figure 2 which shows the amino acid model and gene structure of the human PA R.

Figure 3 which shows a targeted modification of 204D22 DNA to introduce an IRES- LacZ marker gene; Figure 4 which is a photographic image;

Figure 5 which is a photographic image;

Figure 6 which is a photographic image; and

Figure 7 which is a photographic image.

In slightly more detail:

Figure 1 shows the complete sequence of the human PACiR gene with extensive regions of 5' and 3' flanking sequence has been published in GenBank (Accession no. AC006466: Waterston R.H.; "The sequence of Homo sapiens clone"; Genome Sequencing Center, Washington University School of Medicine, 4444 Forest Park Parkway, St. Louis, MO 63108, USA). AC006466 (in the version published in GenBank on 05-MAR-1999) consists of 184514 bp of DNA sequence consisting of 2 unordered pieces (1-78955 and 78973 -184514). The second of these pieces appears to contain the complete sequence of the human PACiR gene, with extensive regions of 5' and 3' flanking sequence. The location of the 18 exons of the human PACi receptor gene (17 coding exons and one exon 5' of the translated region called 5' UTR) within AC006466 is presented in Figure 1. Exons 14a and 14b differ by 3bp in length and result from the use of alternative splice acceptor sites. The translation initiation codon (ATG) is at 103354-103356. The translation termination codon (TGA) is at 146768-146770.

Figure 2 shows in A, a model of the predicted amino acid sequence for the human PACiR showing seven transmembrane-spanning regions (boxed amino acids), potential sites for N-linked glycosylation (tridents), seven highly conserved cysteine residues (triangles). Numbered arrows indicate regions of the human PACjR encoded by the 17 coding exons of the gene. Amino acid products of the alternatively spliced exons, 13 and 14, are shown in brackets with their location in the third intracellular loop indicated by the arrow.

Figure 2 shows in B, a model of the gene structure of part of the the human PACiR showing the consensus sequences for the intron-exon splice sites for exons 13 and 14. Two consecutive splice sites for the exon 14 give rise to two possible splice variants. The information provided is based on Pisegna and Wank (1996) with additional information supplied by the inventors.

Figure 3 shows a recombination cassette which was constructed by cloning a LacZ reporter gene, downstream of an IRES, between sequences (A', B') from the target gene that flank the desired integration site. Region A' is a 0.6 kb Bam HI- Hind III fragment of the hPACiR gene containing the stop codon; region B' is a 1.6kb Hind III - Sst I fragment of the hPACiR gene immediately downstream of A'. Corresponding sequences in the PAC 204D22 are shown as A and B. The recombination cassette is introduced into a shuttle vector containing the E. coli RecA gene (II). The origin of replication for the shuttle plasmid (Ori-ts) is temperature sensitive, so that it will replicate in cells growing at the permissive temperature (30°C) but not in cells growing at the restrictive temperature (42 - 44°C). The shuttle vector is transformed into E. coli containing the PAC 204D22 (I) and transformants containing both the shuttle vector and the PAC are grown overnight at 30°C in the presence of both tetracycline (carried by the shuttle vector) and kanamycin. Because the shuttle vector carries the RecA gene, homologous recombination can occur between the shuttle vector and the PAC. At 42°C, only cells in which homologous recombination has occurred can survive in the presence of tetracycline. Two possible recombination events can occur giving rise to alternative products (Ilia and Illb). In either case, a further homologous recombination event (IVa and IVb), leading to the isolation of the desired recombinant PAC (V), can be selected for by growth at 37°C in the presence of kanamycin and of fusaric acid (which selects for the loss of tetracycline resistance gene encoded by the shuttle vector).

Figure 4 (a,c) shows a histochemical staining of the LacZ reporter gene in a transgenic mouse embryo (El 1.5) expressing a PAC containing the human PACiR gene, (b) shows the in situ hybridization of the endogenous PACiR gene in a El 2.5 mouse embryo. The striking similarity between the expression patterns of the LacZ reporter gene and the endogenous PACiR gene can be readily observed.

Figure 5 (A-F) shows the distribution pattern of LacZ expression in coronal sections of adult transgenic mouse brain. The abbreviations used in (A-F) are detailed as follows: ad, anterodorsal thalamic nucleus; aha, anterior hypothalamic nucleus; amg, amygdala; aon, anterior olfactory nucleus; av, anteroventeral thalamic nucleus; eg, cingulate cortex; dg, dentate gyrus; fr, frontal cortex, pir, piriform cortex; pvt, paraventricular nucleus of the thalamus, re, reuniens thalamic nucleus.

Figure 6 shows the distribution pattern of LacZ expression in: (A) Coronal section of the adult brain, showing LacZ expression in the granule cells of the cerebellum; (B) the pituitary gland: staining is strongest in a population of cells in the anterior lobe of the pituitary (ap), and is absent from the neurointermediate lobe (nil); (C) section through the cervical spinal cord showing expression of LacZ in the superficial laminae of the dorsal horn (arrowed).

Figure 7 shows the developmental defects of 9.5-day transgenic embryos. Embryos were obtained from interbreeding of heterozygous transgenic mice A 149 and stained with X- gal. (A) a transgenic embryo with no gross defect (probably heterozygous for the transgene). (B-D) three embryos out of 8 littermates displayed retarded forebrain development and a failure of the cranial neural folds to converge and fuse at the dorsal midline.

Example 1

Isolation of PAC clones encoding the hPACiR gene

PAC clones encoding the hPACiR gene were obtained by hybridization of the RPCI-1 human PAC library (loannou and de Jong, 1996) with full length rat PACiR cDNA. The structures of positive clones were analysed by Southern blotting of restriction digests and by PCR using primers from hPACiR cDNA that were predicted to flank intron sequences. Targeted modification of a PAC clone encoding the hPACiR gene to introduce an IRES-£αcZ marker gene

An adaptation of recently published methods for the modification of BACs by homologous recombination (Yang et al, 1997) was used to introduce a LacZ reporter gene into the 3' untranslated region of the hPA R gene in the PAC clone 204D22 (Figure 3).

Two overlapping restriction fragments of PAC clone 204D22, containing the translation stop codon of the human PACiR gene, were isolated and cloned into pBluescript SK" in such an orientation that the T3 promoter of the vector was adjacent to the 5' end of each fragment. Clone p3'B2 contained a 4.5 kb Bam HI fragment of 204D22 DNA cloned into the Bam HI site of pBluescript SK" and clone pH39 contained a 0.7 kb Hind III fragment of 204D22 DNA (region A, Fig. 3), cloned into the Hind III site of pBluescript SK".

A 3.5 kb cassette containing the LacZ gene and SV40 polyadenylation sequences was isolated from pMRβ-ZαcZ-PA (Shen et al, 1991) by Sal I (complete) and Eco Rl (partial) digestion and inserted into the Sal I-Eco Rl sites of pBluescript SK", generating pSK"

LacZ-? A. The IRES was introduced into pSK"ZαcZ-PA by replacing the 1.1 kb Xba I (in the polylinker) - Eco RV (in the LacZ sequence) fragment with a 1.7 kb Xba I-Eco RV fragment (IRES-5'-ZαcZ) from pIRES-bgeo (Mountford et al, 1994), resulting in pIRES- LacZ-?A.

An Xba I (blunt ended) - Sal I fragment of pIRES-Z cZ-PA was cloned into the (blunt ended) Cla I and Sal I sites of pH39, to generate pH39ZαcZ. The 1.2 kb RecA gene from pSVLRec (Yang et al, 1997) was excised with Bam HI, blunt-ended with Klenow and cloned into the Eco RV site of pBluescript SK" (pSK'RecA). A 1.6 kb Hind III - Sst I fragment of p3 'B2 containing hPACiR sequences downstream of those in pH39 (region B, Fig. 3) was isolated, blunt ended with Klenow, cloned into the Eco RV site of pBluescript SK", re-excised by digestion with Bam HI and Hind III (blunt ended) and cloned into the Bam Hl-Sma I sites of pSK"RecΛ (p3 ΗPR-Rec ). A Not I-Bgl II fragment of pH39Z cZ was cloned between the Not I-Bam HI sites of p3ΗPR-Rec . Finally the recombination cassette was isolated by Bam Hi-Sal I digestion and cloned into Bam Hi-Sal I sites of the temperature-sensitive pSVl. RecA vector (pSVl-PR-ZαcZ-PR- RecA: construct II in Figure 3).

The circular (pSVl-PR-ZαcZ-PR-Rec ) DNA was introduced into the PAC clone 204D22. Two classes of homologous recombinants (Ilia and Illb in Figure 3) were identified by Southern blotting, using probes corresponding to regions A and B (Figure 3), of Eco Rl + Bam HI digests of PAC clones that grew at 42°C in the presence of tetracycline and kanamycin. A further round of homologous recombination was achieved by growth of recombinants of class Ilia at 37°C in the presence of kanamycin and fusaric acid, resulting in a PAC clone in which IRES-ZαcZ sequences were inserted at a Hind III site which is 100 bp downstream of the stop codon of the hPACiR gene (PACiR-ZαcZ: construct V in Figure 3). The desired recombinant was again identified by Southern blotting, using probes corresponding to regions A and B (Figure 3), of Eco Rl + Bam HI digests. Not I digested PA R-ZαcZ DNA (construct V) was injected into fertilised eggs.

LacZ staining of transgenic mouse embryos expressing the PACiR-ZαcZ construct

10.5 - 11.5 day mouse embryos were rinsed in phosphate-buffered saline (PBS) and fixed for 20 minutes in 1% formaldehyde, 0.2% glutaraldehyde, 0.02% NP-40 in PBS at 4°C. Embryos were washed twice (15 minutes each) in 2mM MgCl2, 0.01% Sodium deoxycholate, 0.02% NP-40 in PBS, then transferred to X-Gal staining solution Clmg/ml X-Gal, 5mM K4Fe(CN)6, 5mM K3Fe(CN)6, 2mM MgCl2, 0.01% Sodium deoxycholate and 0.02% NP-40 in PBS) and incubated (except where specified) overnight at 30°C. Stained embryos were washed in PBS and examined directly or after clearing in 40% and 80% glycerol in PBS. LacZ staining of adult transgenic mice expressing the PAC]R-ZαcZ construct

Mice were killed with a lethal dose of sodium pentobarbitone and briefly perfused through the heart with PBS to remove blood followed by a longer perfusion with approximately 50ml of an ice-cold fixative solution (4% paraformaldehyde in PBS pH 7.4). The brains and internal organs were dissected rapidly and post-fixed in the same fixative for 1 hour at 4°C. Subsequently, the brain was transferred to 30% sucrose in PBS overnight at 4°C and 40 μm coronal sections were cut on a freezing microtome. The sections were washed in PBS, transferred into X-Gal staining solution (see above) and incubated with gentle shaking at 30°C for 2 hours. After staining had developed, sections were transferred into PBS and mounted onto slides before examination and photography. Other tissues were stained without sectioning in a manner identical to that described for embryos above. Optionally, stained tissues were equilibrated with 30% sucrose in PBS before the preparation of sections as described above.

RESULTS 1

Characterization of PAC clones containing human PACiR gene

Two overlapping PAC clones (204D22 and 221D1) encoding the hPACiR gene were identified in the RPCI-1 human PAC library (loannou and de Jong, 1996). PCR analysis using primers flanking the predicted intron sequences indicated that PAC 204D22 contained the entire PACiR gene (~50kb). Restriction mapping of 204D22 DNA with Bss HII, Mlu I, Sfi I and Sal I indicated that it contained in addition approximateLy 70 kb of upstream sequence and approximately 10 kb of downstream sequence. Two STS markers WI-7721 and SWSS1736 were mapped on 224D22 PAC DNA (data not shown).

Generation of transgenic mice co-expressing hPACiR and IRES-ZαcZ reporter genes

An IRES-ZαcZ cassette, flanked by two genomic fragments (0.6 and 1.6 kb) of the hPACiR gene either side of the stop codon, was subcloned into a temperature-sensitive pSVl.RecΛ vector (pSVl-PR-ZαcZ-PR-i?ec ). After transformation of pSVl-PR-ZαcZ- ?R-RecA DNA into the 204D22 PAC clone, correct homologous recombinants with IRES-ZαcZ sequences integrated lOO bp downstream of the stop codon of the hPA R gene (PACiR-ZαcZ), were identified by Southern blotting (data not shown). Not I digested PACiR-ZαcZ DNA was injected into fertilised eggs. Sixty-seven offspring were obtained after embryo transfer and 15 transgenic founders were identified by PCR.

The size of transgene in each founder was characterized using 5 additional pairs of PCR primers: Wi-7721 for the extreme 5^' and 3 T7END for the extreme 3' of the PAC DNA, SWSS1736 and HPR5' close to the promoter region of the hPACiR gene, and ZαcZ primers to detect the presence of the reporter gene. Nine founder animals contained the entire PAC DNA injected, 4 were truncated by <10 kb at the extreme 3 ' end (after the ZαcZ gene), one lacked the 5' end of the hPACiR gene, and one appeared to contain PAC DNA that had not been modified by insertion of the IRES-ZαcZ cassette. Transmission of the intact PAC DNA was obtained from 6 independent founder animals.

LacZ staining of transgenic mouse embryos expressing the PACχR-ZαcZ construct

Embryonic expression of the transgene was examined in 15 independent transgenic lines.

Table 1: PAC 204D22/IZ Transgenic Mice

Analysis of 15 independent transgenic lines containing the PAC clone PACiR-ZαcZ. Each line was analysed for the presence (+) or absence (-) of 6 marker DNA sequences present in clone PACiR-ZαcZ for transmission (ie production of transgenic offspring from the founder animals) and for expression (assessed by LacZ staining) in embryonic and adult tissues). Expression levels are given on an arbitrary scale, where (+) denotes detectable expression and (++++) denotes very high levels of expression. In line A.149.3, very weak expression was seen only in the embryonic heart. In line 162, only part of the PACIR gene and LacZ reporter gene was integrated: in this line, there was LacZ expression in all tissues of the embryo (constitutive expression). The markers were as follows:

Marker 1 is WI-7721 as defined in the Genome Database (The Johns Hopkins University School of Medicine, Baltimore, Maryland: http://gdbwww.gdb.org). As described in that database, WI-7721 is identified by PCR using primers WI-7721-1 (5'-CTGCCTCATCACGCCACT-3') and

WI-7721 -2 (5 ' -T AC AGGTTTATTGGCTCCTCTG-3 ' ).

Marker 2 is sWSS1736 as defined in the Genome Database (The Johns Hopkins University School of Medicine, Baltimore, Maryland: http://gdbwww.gdb.org). As described in that database, sWSS1736 is identified by PCR using primers

sWSS 1736a (5"-TCACATTTACCAGAAACC-3') and

sWSS1736b (5'-GTAAATAATGGGGATAGG-3')

Marker 3 is a 329bp sequence from the intron separating the 2nd and 3rd coding exons of the PACiR gene, defined by PCR primers

549521 (5'-ACTATGGTGACTATGACAGACC-3') and

549522 (5'-TCACCTGCTTGTTGAACTCTGG-3').

Marker 4 is a 389bp sequence from the intron separating the 14th and 15th coding exons of the PACiR gene, defined by PCR primers

549523 (5'-AGTTGGAGATTGCCGATGCC-3') and

549524 (5'-TCAGTCAATAGCCTGTAGAACC-3').

Marker 5 is a 657bp sequence from coding region in of the E. coli LacZ gene, defined by PCR primers

574511 (5'-TAGGTAGTCACGCAACTCGC-3^ς) and

574512 (5'-TGGCGTAATAGCGAAGAGGC-3^ς). Marker 6 is a 137bp of genomic DNA sequence from the human PACIR gene 4212bp downstream of the stop codon, defined by PCR primers

549519 (5'-ATTCTGTGTCCATCTGAGCC-3') and

549520 (5 '-GGATGAAGACTGCTGGAAGG-3').

From Table 1, it can be seen that the 9 transgenic lines are: A149.1, A162.2, A162.5, A162.6, A162.7, A149.2, A162.1, A149.3, A162.

With exception of lines A149.3 and A162, all lines expressed the ZαcZ gene in a pattern strikingly similar to that of the endogenous mouse PACiR gene (Sheward et al, 1998; Sheward et al, 1996; Waschek et al, 1998). Intense ZαcZ staining was observed in the roof and floor plates of the neural tube, the hindbrain, midbrain and diencephalon and was present also in the dorsal root ganglia (Figure 4).

LacZ expression in adult transgenic mice

The expression of the transgene was examined in adult mice from 6 independent transgenic lines. With exception of one line (A 162), all lines expressed the ZαcZ gene in a pattern that closely mimicked that of rodent PA R mRNA (Hashimoto et al, 1993;

Hashimoto et al, 1996; Shioda et al, 1997; Spengler et al, 1993) and protein (Shioda et al, 1997), with widespread expression in the CNS (Figures 5 and 6). There was especially high expression in the hippocampus, where the most intense ZαcZ staining was observed in granule cells of the dentate gyms; high expression was also seen pyramidal cells in the CA1 - CA3 regions. Other prominent sites of expression included the piriform, cingulate and frontal cortices, the hypothalamus (especially the anterior hypothalamic area), many thalamic nuclei (including the anterodorsal, anteroventral, paraventricular and reuniens nuclei), the anterior olfactory nucleus and the external granule cell layer of the cerebellum. In the spinal cord, there was strong ZαcZ staining throughout the superficial laminae of the dorsal horn, most intense adjacent to the entry point of the dorsal roots. Staining was present in a population of cells in the anterior pituitary gland, but was absent from the neurointermediate lobe. There was no staining in sections of brain from non-transgenic control mice.

Although there were no gross developmental defects in herozygous transgenic mice, a failure of the cranial neural folds to converge and fuse at the dorsal midline and retarded forebrain development were observed in a portion of 9.5-day transgenic embryos from interbreeding of heterozygous transgenic mice of lines A149, A149.2 and A162.5. This result indicates that studies of transgenic mice expressing the human PACl receptor may contribute to our understanding of disorders of human brain development and may suggest new approaches to their prevention and/or treatment .

EXAMPLE 2

Identification of splice variants of the PACiR

The high levels of expression of the human PACl receptor in the transgenic mouse embryo was exploited to identify splice variants of the receptor. Total RNA was isolated from three independent lines of 9.5 - 11.5 day transgenic mouse embryos using RNAzol™ B (Biogenesis Ltd, Poole, Dorset) and cDNA was synthesised using the Marathon™ cDNA amplification kit (Clontech). The entire coding region of the human PACl receptor cDNA was amplified by PCR using either primers 604738 and 633776 or primers 604738 and 633777 (Table 2). Subsequently, the existence in human brain of novel splice variants found in transgenic mouse embryos was investigated by PCR of cDNA from human brain hippocampus (HBH), cortex (HBC), striatum (HBJ5), and human fetal brain (HFB, 16-48 weeks, Life Technologies).

Table 2: PCR primers used

604738 (5 ' -GACCTGCCGCTGCTGTC AGTGG-3 ' )

633776 (5 ' -CTC AGGTGGCC AG ATTGTC AG-3 ' )

633777 (5'-TGGATGGAGGAGAGGAGGAGGAGG-3') 604739 (5'-CCTGGCTGCTCTCCTCCTGC-3')

604744 (5 ' -CTGACC AGGACC ATCTC ACC-3 ' )

604743 (5'-GTGAGATGGTCCTGGTCAGC-3')

604746 (5'-TTCCGACCAGCCATCCTCCG-3')

604781 (5 '-TTGATGACACAGGCTGCTGG-3')

604789 (5 '-AACAGTAGAGAACAGCC ACC-3')

604742 (5 ' -CTGGAGAGGAATCATTGAAGCC-3 ')

604776 (5'-GCTGTAGCCAACCGTGTAGAGG-3')

604778 (5 '-TGTCCTGCTCCGCATACAGAATCC-3')

604780 (5 ' -CGATG AAC AGCC AGAAGT AGTTGG-3 ' )

PCR products were cloned into pGem®-T Easy (Promega) and characterised by sequencing and by PCR using primer pairs 604738 + 604744 (amplifies exons 1 - 3), 604743 + 604746 (amplifies exons 3 - 6), 604739+ 604746 (amplifies exons 1 - 6) and 604781 + 604789 (amplifies exons 11 - 16).

RESULTS 2

133 independent clones were characterised and 14 different isoforms of the PACiR were identified as shown in Table 3. Analysis of 381 independent clones obtained by PCR of human brain cDNA confirmed the presence of 10 of these isoforms, together with three more not identified in transgenic mice. Our data indicates that up to 32 distinct splice variants of the PAC receptor may exist. Isoforms 25 - 32 have not been described previously and are of particular interest since they have a non-functional exon 3. These isoforms lack exon 3 which encodes a region of the gene which is thought to be necessary for ligand-activated signal transduction. Within the amino acid sequence encoded by exon 3 is an aspartate residue (Asp59) homologous to a residue (Asp60) in the sequence of the mouse receptor for growth hormone releasing hormone (GHRH) which is mutated to glycine in the little strain of dwarf mice (Godfrey et al., 1993; Lin et al, 1993). This amino acid substitution results in a receptor incapable of responding to GHRH.

Isoforms 9 - 16, containing exons 3 and 5 but with a non-functional exon 4. These isoforms which lack exon 4 have not been described previously and may have novel pharmacological properties since exon 4 is in a region of the PACjR which has been shown to influence the relative affinity of the receptor for PACAP-38, PACAP-27 and VIP.

Isoforms 7, 15, 23 and 31 (with non-functional exons 13, 14 and 15) have not been described previously. These isoforms lack 13, 14 and 15. Isoforms 8, 16, 24 and 32 (with non-functional exons 13, 14, 15 and 16) have also not been described previously. These isoforms lack exons 13, 14, 15 and 16. These isoforms are of particular interest since they encode truncated receptors with 5 transmembrane domains rather than the 7 of the full-length receptor. These isoforms may be impaired in their ability to respond to PACAP by coupling to intracellular signalling pathways.

Table 3: Splice variants of the human PACi receptor identified in transgenic mouse embryos

isoform Exons present in transcript number of clones isolated

1 + 2 3 4 5 6 - 12 13 14a 14b 15 16 17 Al Al A HBH HBC HBS HFB

49 49 1

2 6

2. 5

1 X X X X X X

2 X X X X X X

3 X X X X X 4 1

4 X X X X X 4 3

5 X X X X X 4 5 3

6 X X X X 34 4 2 60 59 51 24

7 X X X

8 X X 1 8

9 X X X X X X X

10 X X X X X X X

I I X X X X X X

12 X X X X X X

13 X X X X X X

14 X X X X X 4 1 20 10

15 X X X X 1

16 X X X 1

17 X X X X X X X X

18 X X X X X X X X

19 X X X X X X X 1 3 0 X X X X X X X 1 5 1 X X X X X X X 3 2 X X X X X X 45 5 2 ^■> 26 14 37 3 X X X X X 1 6 8 4 X X X X 3 3 5 X X X X X X X 6 X X X X X X X - 7 X X X X X X 8 X X X X X X 3 9 X X X X X X 0 X X X X X 12 3 1 12 5 10 1 X X X X 2 X X X 1 EXAMPLE 3

Cell lines expressing the novel isoforms (7 - 16, 23 - 32) of the PACi receptor.

cDNA encoding novel isoforms of the PACl receptor are transfected into cell lines to determine whether the receptor responds to PACAP or to any other ligand or if the receptor is constitutively active.

cDNA encoding novel isoforms of the PAC 1 receptor are co-transfected with cDNA from other splice variants of the receptor into cell lines to determine whether the novel isoforms potentiate or inhibit the action of PACAP acting through other splice variants.

Cells from transgenic animals expressing one or more isoforms are also grown in primary culture to determine their mechanism of action.

RESULTS 3

Our results indicate that PAC receptor isoforms with non-functional exon 3 or exon 15 may be unresponsive to PACAP.

These PACi receptor isoforms have either dominant negative function (interfering with the action of PACAP when expressed together with other splice variants) or they are constitutively active (activating intracellular signalling pathways without any requirement of PACAP binding).

Our results demonstrate that PACi receptor isoforms with a non-functional exon 4 may differ from other isoforms in their responsiveness to PACAP or other ligands. EXAMPLE 4

Definition of the transcriptional start site of the human PAC 1 receptor gene

To define the transcriptional start site of the human PACl receptor gene, we amplified the 5' end of full length PACi receptor cDNA (prepared as above using the Marathon™ cDNA amplification kit) using the Advantage® cDNA PCR kit (Clontech). Six independent PCR reactions were used, using adaptor primer 1 (supplied with the Marathon™ cDNA amplification kit) together with primers from exons 3 (604742), 4 (604744), 7(604746), 8 (604776), 9 (604778) and 10 (604780) of the human PACl receptor gene (Table 2).

A nested PCR was performed on these products using adaptor primer 2 (supplied with the

Marathon™ cDNA amplification kit) and a primer (686297) corresponding to a sequence within the first coding exon of the human PACi receptor gene. PCR products were separated by agarose gel electrophoresis, purified using the Wizard™ PCR Preps DNA

Purification System (Promega), cloned into pGem®-T Easy (Promega) and characterised by sequencing.

The 5' ends of all the clones lie within the region presented as SEQ ID No 3. The sequence of the longest of the clones identified is underlined in SEQ ID No 3.

Our sequence data provides further sequence data extending 143bp upstream of the translation initiation (ATG) codon. The region upstream of the transcription start site of the human PACi receptor (SEQ ID No 3) contains DNA sequences that are the binding sites for transcription factors that regulate the expression of the PACiR gene. Further analysis of this region allows the identification of such factors and permits the identification of treatments capable of regulating gene expression in vivo. Such strategies for the treatment of human disease can be easily tested in PAC receptor-ZαcZ transgenic mice. SUMMARY

In the present invention, transgenic mice are provided that express a construct encoding the human PAC R gene. The transgenic mice were created using a PAC construct containing the human PACiR gene flanked by extensive regions of 5' and 3' flanking sequence and with a ZαcZ reporter gene inserted into the 3 ^' untranslated region of the gene, downstream of an IRES (Mountford et al, 1994).

In transgenic mice comprising the PAC construct of the present invention, ZαcZ was expressed in a tissue-specific and developmentally regulated pattern that closely mimics that of the endogenous mouse PACiR. Consistent with previous studies using in situ hybridization (Hashimoto et al, 1996; Shioda et al, 1997), the most prominent site of expression of the ZαcZ reporter gene in human PACiR-ZαcZ mice was the hippocampus, most intensely in the dentate gyms.

The present invention demonstrates that the PACiR is present in the dorsal horn of the spinal cord. These data suggest that PACAP released from C-fibres in the spinal cord may play a role in modulating nociception.

Studies of the regulation of human PACiR gene expression in transgenic animals facilitates the understanding of the functions of the PACiR gene in the developing and adult nervous system. In addition, studies of the regulation of PACi R gene expression in hPAC 1 R-ZαcZ mice in vivo and in vitro, using ZαcZ reporter gene expression as a simple assay are adaptable to a high throughput screening assays to facilitate the identification of agents capable of acting as PACiR agonists and antagonists when administered to human subjects. Moreover, breeding of hPACiR-ZαcZ mice with PACiR knockout mice, can provide humanised" animals expressing the PACiR with human pharmacology, permitting the evaluation of the effectiveness of PACl R agonists and antagonists when administered to human subjects.

In another aspect, the present invention also demonstrates that up to 32 distinct isoforms/splice variants of the PAC] receptor may exist. In this respect: (i) PACl receptor isoforms 25 - 32 have not been described previously and are of particular interest since they lack exon 3. Exon 3 may encodes a region of the gene which is thought to be necessary for ligand-activated signal transduction. This group of isoforms includes a splice variant of the PACiR gene called isoform 30 which was designated in UK patent application number 9909446.8 as isoform 18). Studies subsequent to the filing of UK patent application number 9909446.8 by Dautzenberg et al (1999: J Neuroendcrinol 11 : 941-949) have confirmed the existence of isoform 30 (designated in UK patent application number 9909446.8 as isoform 18) and have shown that this isoform has a greatly reduced ability to respond to ligands such as PACAP.

(ii) PACl receptor isoforms 9 - 16, comprising exon 3 and exon 5 but lacking exon 4 have not been described previously. These isoforms may have novel pharmacological properties since exon 4 is in a region of the PACiR which has been shown to influence the relative affinity of the receptor for PACAP-38, PACAP-27 and VIP.

(iii) PACl receptor isoforms 7, 15, 23 and 31 lacking exons 13, 14 and 15 and isoforms 8, 16, 24 and 33 lacking exons 13, 14, 15 and 16 have not been described previously. These isoforms are also of particular interest since they encode truncated receptors with 5 transmembrane domains rather than the 7 of the full-length receptor. These isoforms may be impaired in their ability to respond to PACAP by coupling to intracellular signalling pathways.

Studies of the regulation of PAC} R isoform/splice variant gene expression in transgenic animals facilitates the understanding of the functions of the PACiR isoform/splice variant gene in the developing and adult nervous system. In addition, studies of the regulation of PACiR isoform/splice variant gene expression in hPACiR-ZαcZ mice in vivo and in vitro, using ZαcZ reporter gene expression as a simple assay are adaptable to high throughput screening assays to facilitate the identification of agents capable of acting as PAC R agonists and antagonists when administered to human subjects. Moreover, breeding of hPACiR isoform splice variant-ZαcZ mice with PACiR knockout mice, can provide humanised" animals expressing the PAC R with human pharmacology, permitting the evaluation of the effectiveness of PAC l R agonists and antagonists when administered to human subjects.

All publications mentioned in the above specification are herein incoφorated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

SEQUENCE LISTING FREE TEXT PART OF THE DESCRIPTION

SEQ ID No 1 : presents the cDNA sequence of human PACl receptor cDNA - the coding region is underlined.

SEQ ID No 2: presents the human PACiR protein sequence.

SEQ ID No 3: presents the sequence of the putative promoter and first (untranslated) exon of the human PAC receptor gene.

SEQ ID No 4: presents a nucleotide sequence encoding an internal ribosomal entry site (IRES).

SEQ ID No 5: presents a nucleotide sequence encoding an internal ribosomal entry site (IRES).

SEQ ID No 6: presents the amino acid sequence (single letter nomenclature) of the protein encoded by isoform 30 (designated in UK patent application number 9909446.8 as isoform 18).

SEQ ID No 7: presents the nucleotide sequence of the protein coding region of isoform 30 (designated in UK patent application number 9909446.8 as isoform 18).

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Claims

1. A transgenic organism comprising a PAC vector.

2. A transgenic organism according to claim 1 wherein the PAC vector comprises a

PACR gene, a reporter gene and an element capable of causing co-expression of the PACR gene and the reporter gene.

3. A transgenic organism according to claim 2 wherein the PACR gene is a human PACiR gene.

4. A transgenic organism according to claim 3 wherein the human PA R gene comprises the sequence presented as SEQ ID No 1 or a variant, homologue, fragment or derivative thereof.

5. A transgenic organism according to claim 4 wherein the human PA R gene encodes a protein comprising the sequence presented as SEQ ID No 2 or a variant, homologue, fragment or derivative thereof.

6. A transgenic organism according to claim 5 wherein the human PA R gene encodes a human PACiR receptor which has at least a non-functional exon wherein the non- functional exon is selected from the group consisting of exon 3, exon 4, exon 13, exon 14, exon 15 and exon 16 or combinations thereof, preferably exon 3.

7. A transgenic organism according to claim 6 wherein the human PACjR gene comprises the sequence presented as SEQ ID No 7 or a variant, homologue, fragment or derivative thereof.

8. A transgenic organism according to claim 7 wherein the human PACiR gene encodes a protein comprising the sequence presented as SEQ ID No 6 or a variant, homologue, fragment or derivative thereof.

9. A transgenic organism according to claim 2 to claim 8 wherein the reporter gene is capable of producing a visually detectable signal.

10. A transgenic organism according to claim 2 to claim 9 wherein the reporter gene is capable of producing an immunologically detectable signal.

11. A transgenic organism according to claim 2 wherein the element is an internal ribosomal entry site (IRES).

12. A transgenic organism according to claim 1 1 wherein the IRES has a nucleotide sequence which comprises the sequence presented as SEQ ID No 4 or a variant, homologue, fragment or derivative thereof.

13. A transgenic organism according to claim 11 wherein the IRES has a nucleotide sequence which comprises the sequence presented as SEQ ID No 5 or a variant, homologue, fragment or derivative thereof.

14 A transgenic organism according to any one of the preceding claims wherein the expression of the reporter gene is under the control of a regulatory sequence from the human PA R gene.

15. A transgenic organism according to claim 14 wherein the regulatory sequence comprises the sequence presented as SEQ ID No 3 or a variant, homologue, fragment or derivative thereof.

16. A transgenic organism comprising a PACR gene.

17. A transgenic organism according to claim 16 wherein the transgenic organism is capable of co-expressing a PACR gene and a reporter gene such that the expression pattern of the PACR gene can be determined by measuring a detectable signal produced by the reporter gene.

18. A transgenic organism according to claim 16 or claim 17 wherein the transgenic organism is capable of co-expressing a human PA R gene.

19. A transgenic organism according to claim 18 wherein the transgenic organism is capable of co-expressing a novel isoform of a human PA R gene.

20. A transgenic organism according to claim 19 wherein the novel isoform of a human PACiR gene is selected from the group of isoforms consisting of: isoforms 25-32; isoforms 9-16; isoforms 7, 15, 23 and 3; isoforms 8, 16, 24, and 32; or combinations thereof, preferably isoform 30.

21. An isolated isoform of the human PACiR gene wherein the isolated isoform is selected from the group consisting of: isoforms 25-32; isoforms 9-16; isoforms 7, 15, 23 and 3; isoforms 8, 16, 24, and 32; or combinations thereof, preferably isoform 30.

22. A transgenic organism according to any of the preceding claims wherein the transgenic organism is a PAC transgenic organism.

23. A transgenic organism according to claim 22 wherein the transgenic organism is a PAC transgenic mouse.

24. A transgenic organism according to claim 23 wherein the transgenic organism is a PAC transgenic mouse comprising a human PACiR gene or an isoform/splice variant thereof.

25. A transgenic organism according to claim 24 wherein the isoform/splice variant is an isoform of the human PACiR gene selected from the group consisting of: isoforms 25- 32; isoforms 9-16; isoforms 7, 15, 23 and 3; isoforms 8, 16, 24, and 32; or combinations thereof, preferably isoform 30.

26. A transgenic organism according to claim 25 wherein the isoform is identified by a probe presented in Table 2.

27. A transgenic organism according to claim 26 wherein the isoform is identified by a group of primer pairs selected from a group consisting of 604738 + 633776; 604738 + 633777; 604738 + 604744; 604738 + 604746; and 604738 + 604789; 604743 + 604746; 604739 + 604746; and 604781 + 604789.

28. A nucleotide sequence comprising at least 10 contiguous nucleotide bases derived from the human PA R gene sequence as defined in claim 7 or from any one or more of the isoforms of the human PACjR gene as defined in claim 20 or claim 21 or claim 25 or claim 26 for use as a probe.

29. A method for preparing a transgenic organism comprising providing (such as preparing) a PAC vector and inserting said vector into a transgenic organism thereby to form said transgenic organism.

30. A method for preparing a transgenic organism according to claim 29 wherein the transgenic organism is a transgenic mouse.

31. An assay method for identifying an agent that can affect the expression pattern of a PACiR gene or the PA R activity thereof, wherein the assay method comprises:

(i) administering an agent to a PAC transgenic organism as defined in claim 19 or a PAC transgenic mouse as defined in claim 23 or claim 24; and

(ii) determining whether the agent modulates (such as affects the expression pattern or activity) of the PACiR gene or the PACiR activity by means of a detectable signal.

32. An assay method according to claim 31 wherein the assay is to screen for agents useful in the treatment and/or modulation of disturbances in any one of: stroke and other cerebovascular disorders; cardiovascular disease; leishmaniasis; immunosuppressive disorders; nociception; and learning and memory functions.

33. An agent identified by the method of claim 32.

34. A process comprising the steps of:

(a) performing the assay according to claim 31 or claim 32;

(b) identifying one or more agents that affect the expression pattern of the PACiR gene or the PACiR activity thereof;

(c) preparing a quantity of those one or more identified agents.

35. A process comprising the steps of:

(a) performing the assay according to claim 31 or claim 32;

(c) preparing a pharmaceutical composition comprising one or more identified agents.

36. A process comprising the steps of:

(a) performing the assay according to claim 31 or claim 32;

(c) modifying one or more identified agents to cause a different effect on the expression pattern of the PACiR gene or the PACiR activity thereof.

37. Use of a PAC and/or a PACiR gene to study gene function and regulation in a transgenic organism.

38. Use of a PAC and/or a PACiR gene to facilitate the understanding of the functions of the PACiR gene in the developing nervous system and the adult nervous system.

39. Use of a PAC and/or a PA R gene to screen for agents capable of affecting PACiR activity or the expression pattern of the PACiR gene in a transgenic organism.

40. Use of a PAC and/or a PACi gene to screen for agents capable of affecting the expression pattern of the human PA R gene in a transgenic organism wherein the expression pattern of the human PACiR gene in the central nervous system (CNS) is in a tissue-specific and developmentally regulated pattern that closely mimics that of the endogenous mouse PACiR gene.

41. Use of a transgenic organism comprising a PAC and/or a PACiR gene to test for potential pharmaceutical and/or veterinary agents.

42. Use of a PAC transgenic mouse according to claim 23 or claim 24 wherein the PAC transgenic mouse is used to evaluate the effectiveness of PA R agonists and antagonists when administered to human subjects.

43. Use of a PAC and or a PACiR gene to screen for agents capable of affecting PACiR expression in the dorsal horn of the spinal cord.

44. Use of an agent in the preparation of a pharmaceutical composition for the treatment of a disorder or condition associated with the expression pattern of a PACjR gene or the PACjR thereof, the agent having an effect on the expression pattern of the PACjR gene or the PACjR activity thereof when assayed in vitro by the assay method according to claim 31 or claim 32.

45. Use of an agent identified by an assay method according to claim 31 or claim 32 in the manufacture of a medicament which affects the expression pattern of a PA R gene or the PA R activity thereof.

46. Use of a PAC vector and/or a PACiR gene and/or expression products thereof in the preparation of a medicament in the treatment and/or modulation of disturbances in any one of: stroke and other cerebovascular disorders; cardiovascular disease; leishmaniasis; immunosuppressive disorders; nociception; and learning and memory functions.

47. Use according to claim 46 wherein the PAC vector and/or a PACiR gene and/or expression products thereof is used to screen for agents that can modulate the activity of the PAC vector and/or PACjR gene and/or expression products thereof.

48. A PACiR agonist wherein the PACiR is the PACiR gene as defined in claim 3 or the expression product thereof.

49. A PACiR antagonist wherein the PA R is the PA R gene as defined in claim 3 or the expression product therof.

50. A PAC mouse.

51. A PAC vector substantially as described herein and with reference to the accompanying Figures.