GB2331752A - Vectors and YACs comprising an IRES - Google Patents

Abstract

YAC vectors are described which may be used individually or in combination to prepare a YAC. A first YAC vector comprises a nucleotide sequence wherein the nucleotide sequence comprises the sequence presented as SEQ ID No.1 or SEQ ID No. 4 or a variant, homologue or derivative thereof. YAC vectors of the invention includes an IRES or a reporter gene (which may be specifically removable). Other vectors claimed include YAC vector modifying vectors which contain an IRES- lacZ-auxotrophic marker casette, and pYAM4 - a vector used to amplify a YAC. Also disclosed are methods of making YAC transgenic animals, and the use of those animals to test for potential pharmaceutical agents.

Description

2331752 VECTORS The present invention relates to vectors, in particular

vectors that are suitable for use as 5 or with or in the preparation of a yeast artificial chromosome.

A yeast artificial chromosome - otherwise known as a YAC - comprises the structural components of a yeast chromosome into which it is possible to clone very large pieces of DNA. By way of example, it is generally possible to clone into a YAC stretches of 1 o DNA that are up to about 1000kb long - which are much larger than when compared with the stretches of DNA that can be cloned into other cloning vectors such as plasmids (typically up to 20kb stretches of DNA), bacteriophage X (typically up to 25kb stretches of DNA), cosmids (typically up to 45kb stretches of DNA) and the P1 vector (typically up to 100kb stretches of DNA) (see Lodish et al 1995 Molecular Cell Biology 3rd 15 Edition, Pub. Scientific American Books, page 233).

YACs were initially proposed by Burke et al (Burke, D.T., G.F. Carle and M.V. Olson (1987) Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 236: 806-812). General introductory teachings on YACs 2o have been presented by T.A. Brown 1995 (Gene Cloning, An Introduction, 3rd Edition, page 325, Pub. Chapman & Hall, pages 139-142).

Typically, a YAC contains the following essential functional elements: a centromere, two telomeres and one or more origins of replication. The centromere is required to 25 correctly distribute the chromosome to daughter cells during cell division; the telomeres are required to ensure correct replication; and the replication origin(s) is (are) present to ensure initiation of DNA replication. The origins of replication are sometimes referred to as ARS elements.

3 However, the use of YACs is by no means limited to mapping of the human genome. For example, the use of YACs has led to the preparation of physical maps of the Drosophila X chromosome containing the shibire (shi) locus (Bliek and Meyerowitz 1991 Nature 351 441). YACs have also been proposed to map plant genomes such as 5 the Arabidopsis genome.

In addition to their usage in genome mapping, YACs have now another important use. In this regard, it has been recently found that under certain conditions YACs can be introduced into mammalian cells (such as murine cells), whereupon they can behave io functionally the same as (or very similar to) endogenous chromosomes. In this regard, it is possible to deliver - such as by use of cell fusion techniques, microinjection techniques or transfection techniques - YACs containing large fragments of human DNA (i.e. the NOI) into the mouse germline. This was initially achieved with a 670kb YAC containing a human X-chromosome fragment (Jacobovitis et al 1993 Nature 362 pages 15 255-258). By way of further example, WO-A-94/23049 reports on a YAC containing the gene coding for P-amyloid precursor protein.

Hence, YACs now enable workers to analyse an or the in vivo functional behaviour of a NOI, such as a human NOI. For these studies, prior knowledge of the sequence and/or 20 function of the NOI need not be necessary.

A review of these in vivo functional studies has been presented by Jacobovitis (Current Biology 1994 vol 4 No 8 pages 761-763), who states:

25 "The ability to replace mouse genes with their human equivalents using yeast artificial chromosome technology provides a powerful new technique for studying the regulation and function of human genes".

For example, the low efficiency with which transgenic animals are produced using YAC DNA (1-5%) compared to DNA from conventional vectors (approximately 10%) is probably caused by the low concentration of YAC DNA available for injection. Assuming that 2 pl of 500kb YAC at a concentration of 1 nglpl is injected into a 5 pronucleus, a fertilised egg receives only 1 molecule of YAC DNA. Amplification of YACs in yeast should provide a possible method for the isolation of more concentrated YAC DNA which should lead to more successful generation of YAC transgenic animals (i.e. a transgenic mammal that comprises a YAC). However, to date, there has not been a totally acceptable solution to this problem.

io For example, Smith et al proposed use of the YAC vector pCGS990 (Smith et al Mammalian Genome 1993 4 pages 141-147). Even though that YAC vector goes some way to overcoming this problem, it nevertheless comprises the TK gene (i.e. the herpes simplex virus thymidine kinase gene) as a selectable marker. This is problematic as 1 5 expression of this gene can cause male sterility in transgenic animals.

Alternatively, or in addition, with current techniques it is not currently possible to readily monitor the in vivo expression pattern of a NOI that has been introduced into an organism - such as a mouse. Current techniques - such as in situ hydridisation, 20 Polymerase Chain Reaction (PCR) and Northern Blotting - are laborious to carry out.

In addition, and by way of further example, none of the earlier reported studies has reported on the co-expression of a NOI and a reporter gene in a YAC.

25 Thus there are problems associated with the known vectors for preparing YACs.

The present invention seeks to improve upon the existing techniques associated with the preparation of and usage of YACs.

30 In this regard, the present invention seeks to provide two types of vectors that can be used on their own or in combination with each other and thereby overcome at least one of the above-mentioned problems.

According to a second aspect of the present invention there is provided one or more of the following embodiments, which for ease have been presented as numbered paragraphs:

5 1. A YAC vector comprising a nucleotide sequence wherein the nucleotide sequence comprises the sequence presented as SEQ ID No. I or SEQ ID No. 4 a variant, homologue or derivative thereof.

2. A vector capable of modifying a YAC or a YAC vector wherein the vector to comprises a nucleotide sequence wherein the nucleotide sequence comprises the sequence presented as SEQ ID No. I or SEQ ID No. 4 or a variant, homologue or derivative thereof.

4. A YAC prepared by the vector according to any one of the abovementioned embodiments.

5. Use of a nucleotide sequence comprising the sequence presented SEQ ID No. I or 20 SEQ ID No. 4 or a variant, homologue or derivative thereof in a vector to prepare a YAC vector or a YAC.

6. Use of a nucleotide sequence comprising the sequence presented SEQ ID No. 1 or SEQ ID No. 4 or a variant, homologue or derivative thereof in a vector to increase the 25 expression efficiency of one or more NOIs within a YAC vector or a YAC.

For convenience, the vector of the first aspect of the present invention is sometimes referred to herein as being an insertion vector; whereas the vector of the second aspect of the present invention is sometimes referred to herein as being an amplification vector.

9 (such as a visually or an immunologically detectable signal) produced by the expression product of the reporter gene.

A YAC transgenic mammal expressing a reporter gene under the control of a 5 regulatory sequence from a human NOI.

Use of a YAC transgenic mammal to test for potential pharmaceutical and/or veterinary agents.

10 An assay method for identifying an agent that can affect the expression pattern of an NOI or the EP ("expression product") activity thereof, the assay method comprising 15 administering an agent to a YAC transgenic mammal according to the present invention; determining whether the agent modulates (such as affects the expression pattern or activity) the NOI or the EP by means of the detectable signal.

An assay method according to the present invention wherein the assay is to screen for agents useful in the treatment of disturbances in any one of: circadian function, sleep disorders, eating disorders, pre-menstural syndrome, autoimmune disorders, birth defects in women and/or sexual dysfunction.

An agent identified by the method according to the present invention.

A process comprising the steps of:

30 (a) performing the assay according to the present invention; 11 or the EP activity thereof when assayed in vitro by the assay according to the present invention.

Use of an agent identified by an assay according to the present invention in the 5 manufacture of a medicament which affects the expression pattern of an NOI or the EP activity thereof.

Use of an agent identified by an assay according to the present invention in the manufacture of a medicament which affects the expression pattern of an NOI or the EP 10 activity thereof.

In accordance with the present invention, at least part of the assay can be carried out in living tissue.

15 A preferred - but non-limiting - example of an NOI is the human serotonin transporter (SERT), preferably the the human serotonin transporter (SERT) presented as SEQ ID No. 2 or a variant, homologue or derivative thereof.

The terms "variant", "homologue", or "derivative" in relation to this aspect of the present 20 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. 2, preferably having at least the same level of activity of the expression product of SEQ I.D. No. 2. In particular, the term "homologue" covers 25 identity with respect to structure and/or function providing the resultant nucleotide sequence has promoter 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.

13 monoclonal, chimeric, single chain, Fab fragments, fragments produced by a Fab expression library and humanised monoclonal antibodies.

The term "expression product" or "EP" means the expressed protein per se but also 5 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.

The term "disturbances in circadian function" means disorders which may lead to impaird physical and mental well-being that can occur through extremes in work patterns 10 such as shift work, in normal ageing, when travelling through time zones (jet lag), and in dementia.

There are a number of advantages associated with the present invention.

15 For example, with the use of the amplification vector it is possible to obtain high copy numbers of YACs.

For example, with the use of the insertion vector it is possible to readily monitor the in vivo expression pattern of a NOI.

Also, with the use of the insertion vector, it is possible to express such as over-express - a NOI and a reporter gene contained within or as a YAC.

It provides a means for producing YAC transgenic animals and the analysis of these 25 animals in terms of expression, regulation and function of NOIs present in YAC DNA.

It facilitates the determination of sites/regions where an NOI is expressed and the identification of agents which may affect the expression pattern of the NOI or the EP activity thereof.

Other advantages associated with the present invention will be apparent from the following text.

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 (mentioned above).

5 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 any suitable organism. In a preferred embodiment, the cell is a to 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).

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

Typically, the cell will be transformed by any one of the following methods: transfection, microinjection, electroporation or microprojectile bombardment, including combinations thereof.

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

For some applications, the transformed cells may be prepared by use of the modified YAC according to the present invention.

The vectors of the present invention can also be used to prepare transgenic organisms that can be used for functional studies of the NOI. 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 NOI in combination with other entities, such as other NOIs, 30 compounds or compositions. In addition, the transgenic organisms can be used to test potential pharmaceutical agents (including veterinary agents).

17 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 5 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 affect 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 1o present on the YAC.

With the present invention, the YAC vector may additionally comprise one or more NOIs. The NOI need not be of known function and/or structure. Preferably, the NOI is of human origin.

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

A review on IRES is presented by Mountford and Smith JIG May 1995 vol 11 No. 5 20 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, WOA94/24301, WO-A-95/32298, and WO-A-96/27676. These references do not disclose or 25 suggest the use of an IRES unit in preparing or being a part of a YAC.

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 30 (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 19 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 5 sequence has promoter 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.

io Sequence identity with respect to any of SEQ ID 1-4 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).

15 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 20 described in detail at http://www.ncbi.nih.gov/BLAST/blast help.html, which is incorporated herein by reference. The search parameters are defined as follows, can be advantageously set to the defined default parameters.

Advantageously, "substantial identity" when assessed by BLAST equates to sequences 25 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.

BLAST (Basic Local Alignment Search Tool) is the heuristic search algorithm employed 30 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.htm]) with a few enhancements. The 21 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 1 o 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 high15 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).

20 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.

30 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 23 In some aspects of the present invention, no gap penalties are used when determining sequence identity.

The present invention also encompasses nucleotide sequences that are complementary to 5 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.

The present invention also encompasses nucleotide sequences that are capable of o 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 15 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 20 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 Tin; 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 30 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.

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 315318), 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 YAC 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.

o 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 YAC libraries, such as YAC human DNA libraries.

20 For example, if the NOI in the YAC 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 25 ability or pattern of that regulatory element.

By way of further example, if the NOI in the YAC has a functional role other than an expression regulatory role then by use of the insertion vector according to the invention workers can fuse (either directly or indirectly such as by means of one or more spacing 3o 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 27 The YAC vector may additionally comprise one or more marker genes. These genes can be chosen from suitable marker genes that are available. An example of a suitable marker gene is PGK-Hyg (see Nara et al 1993 Curr Genet 23(2): pp 134-140).

5 The nucleotide sequence of the present invention can be used to modify a YAC or a YAC vector, such as pYACl, pYAC2, pYAC3 or pYAC4 etc.

The present invention also encompasses combinations of the abovementioned aspects.

i o The following samples were deposited by the MRC Brain Metabolism Unit, Royal Edinburgh Hospital, Morningside Park, Edinburgh, EH 10 5HF in accordance with the Budapest Treaty at the recognised depositary The National Collections of Industrial and Marine Bacteria Limited (NCIMB) at 23 St. Machar Drive, Aberdeen, Scotland, United Kingdom, AB2 1 RY on 24 November 1997 JM 109 pYIV 1 - deposit number NCIMB 40907 JM109 pYIV2 deposit number NCIMB 40908 JM109 pYIV3 - deposit number NCIMB 40909 JM 109 pYIV4 - deposit number NCIMB 40910 20 JM 109 pYAM4 - deposit number NCIMB 40906 The present invention will now be described only by way of example, in which reference shall be made to the following Figures:

25 Figure 1 which is a diagrammatic representation of pYIV I; Figure 2 which is a diagrammatic representation of pYIV2; Figure 3 which is a diagrammatic representation of pYIV3; Figure 4 which is a diagrammatic representation of pYIV4; 29 Figure 14 shows the immunohistochemical staining of the LacZ reporter gene in the suprachiasmatic nuclei of transgenic mice expressing a YAC containing the human VPAC2R gene. The single cell resolution obtainable with the immunohistochemical approach is worthy of note.

Figure 15 illustrates the histochemical staining of P-galactosidase activity in transgenic mice containing the YAC HSC7E526/V 12.

Figures 15a and 15b show staining patterns for R-galactosidase activity in a coronal slice from the brain of a transgenic mouse for whom mouse A108.2 was the father. In (a) the stained suprachiasmatic nuclei are indicated with arrowheads, in (b) an enlarged view is shown. Figure 15c shows staining in the pancreas from the same transgenic mouse (tg) and in a wild type (wt) littermate.

15 Figure 16 illustrates the tissue distribution of P-galactosidase activity in transgenic mice containing the YAC HSC7E526/V 12.

(3-galactosidase (LacZ) activity was determined in tissue extracts from control mice and two independent lines expressing the hVPAC2R-HA-lacZ transgene. Enzyme activity was determined using a chemiluminescent reporter assay system (Galacto-Light Plus, Tropix). ND indicates tissues in which no P-galactosidase activity was detected.

For ease of reference, some parts of the following text has been split according to the vector of the first aspect of the present invention or according to the vector of the second 25 aspect of the present invention.

31 ADE2 gene was isolated from pASZI1, filled in and inserted into the filled in HindIII site of pBluescript SK- in an orientation such that the T7 promoter is adjacent to the 5' end of the ADE2 gene (pSK-ADE2). The IRES-lacZ--PA cassette was released from pIRES-IacZ-PA by Sall digestion, filled in and then cut with NotI. The fragment was 5 inserted into the EcoRI (filled in) and NotI sites of pSK-ADE2, resulting a plasmid (pYIV2) containing and IRES-lacZ-ADE2 cassette (Fig.4). Four unique sites (SacII, NotI, Sall and Xhol) are available for cloning.

pYIV3 For many genes, there is no antibody available to detect the encoded protein. We introduced a haemaggluttinin (HA) epitope tag into pYIV2 so that a commercially available antibody can be used to localise the distribution of the protein product of a YAC transgene within cells. The HA tag was introduced as follows:

The translation stop codon of the human VIP2 receptor cloned in the vector pcDNA3 was converted into an Xhol site by PCR-based mutagenesis. A linker encoding the HA epitope tag flanked by Xhol-XbaI sites 20 5'-TC GAG TAC CCA TAC GAT GTT CCA GAT TAC GCC TCC CTC TAG-3' 3'-ATG GGT ATG CTA CAA GGT CTA ATG CGG AGG GAG ATC AGA TC-5' was cloned into the XhoI-XbaI sites at the end of the VIP2 receptor. The VIP2 receptor25 HA fragment was released from the pcDNA3 vector by BamHI and XbaI (filled-in) digestion and cloned into pBluescript SK- (in which the Xhol site was removed by filling in) atthe Bamf-EcoRV sites generating pSK-VIP2R-HA.

The IRES-lacZ-ADE2 cassette was isolated from pYIV2 with Notl (filled in) and Sall 30 restriction enzymes and inserted into the HindIII (filled in) and Sall sites of pSK-VIP2RHA, yielding a plasmid containing VIM-HA-IRESIacZ-ADE2. The HA-IRES-lacZ- 33 by Schedl et al. (28). Then, plugs were washed in 50 mM EDTA (2 x 30 min) and digested with proteinase K (2mg/ml) in a buffer containing 0.5 M NaCl, 0.125 M Tris pH 8.0, 0.25 M Na2EDTA, 1 % Lithium sulphate, and 0.5 M P- mercaptoethanol at 55 C overnight. Plugs then were washed with TE and stored at 4 C in 0.5 M EDTA.

Pulsed-field gel electrophoresis

DNA plugs were washed in TE (3 x 30min), loaded on a 1 % agarose gel and sealed with 1 % agarose in 0.5 x TBE buffer. Gels were run in 0.5 x TBE buffer at 6V/cm for 24 o hours at 14 C with 60 sec. switch time. After running, gels were stained with ethidium bromide and photographed.

AMPLIFICATION VECTOR 1 5 Construction of pYAM4 pYAM4 was constructed using pYAC4, pBluescript SK- and pBG. pBG is a modification of pCGS990 in which the Sall site has been converted to a Nod site and a PGK-Hyg-loxP cassette has been introduced between the LYS2 and TK genes in 20 pCGS990.

pBG was constructed as follows: The unique Sall site in pCGS990 was converted into Nod with the Xhol-Notl-XhoI linker 5'-TCGAGCGGCCGC-3' 3'-CGCCGGCGAGCT-5' 30 resulting in pCGS990N. A 2044 by Pstl fragment containing the chloramphenicol resistance gene (cm) flanked by two IoxP sites was excised from pUC91ox2cm, blunt ended with T4 DNA polymerase and ligated to the filled-in Sphl site of pHA58 which resuspension in 400 p.1 TE, cells were plated out on SD medium lacking uracil and lysine. Plates were kept at 30 C for 3-5 days. Single colonies were picked and plated onto dishes lacking lysine or tryptophan. Clones which could not grow on the medium lacking tryptophan were selected for further analysis.

Yeast DNA preparation Yeast DNA was isolated with the combined methods of Schedl et al. (26) and Bellis et al. (27). Clones were inoculated into 15 ml of medium (Ura- /Lys-) with 2% of o galactose instead of glucose as the carbon source. When cells had grown to late log phase after 2-4 days, plugs were taken and subjected to novozyme digestion for 4-6 hours as described by Schedl et al. (28). Then, plugs were washed in 50 MM EDTA (2 x 30 min) and digested with proteinase K (2mg/ml) in a buffer containing 0.5 M NaCl, 0. 125 M Tris pH 8.0, 0.25 M Na2EDTA, 1 % Lithium sulphate, and 0.5 M (315 mercaptoethanol at 55 C overnight. Plugs then were washed with TE and stored at 4 C in 0.5 M EDTA.

Pulsed-field gel electrophoresis

20 DNA plugs were washed in TE (3 x 30min), loaded on a 1 % agarose gel and sealed with 1 % agarose in 0.5 x TBE buffer. Gels were run in 0.5 x TBE buffer at 6V/cm for 24 hours at 14 C with 60 sec. switch time. After running, gels were stained with ethidium bromide and photographed.

25 COMBINATION OF INSERTION VECTOR AND AMPLIFICATION VECTOR Combination of the pYIV3 and pYAM4 for YAC transgenic study The vector pYIV3 was used to introduce the haemagglutinin (HA) tag and lacZ reporter 30 gene into two YAC clones,35D8 (500kb) and HSC7E526 (630kb), which contain the human serotonin transporter (SERT) and VIP2 receptor (VIPR2) genes respectively. In order to integrate the lacZ reporter gene into each of the YAC clones by homologous 37 The Notl-Sail fragment of pVHAIZA which contains VIPR2-HA-IRES-IacZ-PA- Ade2 was ligated into NotI-XhoI digested p3'VIPR2, generating a final construct, pLacZVIPR2 +. For efficient homologous recombination in yeast, genomic DNA sequences at least a few hundred by in length must flank the stop codon of the target 5 gene either side of the HA-IRES-IacZ-Ade2 sequences of pYIV3. In pLacZVIPR2+ there is 1.3 kb of VIPR2 genomic sequence upstream and 1.6 kb of VIPR2 genomic sequence downstream of the HA-IRES-IacZ-Ade2 cassette.

Construction of the pLacZSERr vector io The XhoI site in the polylinker of pBluescriptSK- was removed by digesting the vector with XhoI and filling in the recessed 3' termini with Klenow fragment of E. coli DNA polymerase, generating pSKX. A BamHI- Xbal fragment containing the human VIPR2 cDNA with the HA tag at the C- terminus of the coding sequence was subcloned from the 15 pcDNA3 vector into pSKX at the EcoRV site, in an orientation that the 5'end of the cDNA is adjacent to the T3 primer in the pSKX vector, generating pSK-VIPR2-HA. A 5 kb human genomic DNA fragment contaning intron 13 and exon 14 of the SERT gene was cloned into Notl-XhoI sites of pBluescriptSK- using PCR primers 2o 32365 (5'ACT GCA TAG CGG CCG _CAT CTT TCA TTT GCA TCC CC 3') and 32853 (5' TGT GSLCSzA QAG CAT TCA AGC GGA TGT 3') generating pln13. To introduce the HA tag into the C-terminus of the SERT gene product, the 5 kb SERT intron 13 sequence (NotI-XhoI fragment) was used to replace the Notl-XhoI fragment in pSK-VIPR2-HA, generating pInl3- HA. The intron 13 sequence and the HA tag were isolated as a SacIl - Clal (blunt ended) fragment and inserted into 30 SaclI and (blunt ended) NotI sites of pYIV2, generating pIn13-HA-IZA. The sequences downstream of the stop codon in exon 14 of the SERT gene were isolated by PCR of human genomic DNA using primers 39 from the GAL1 promoter interferes with the function of the CEN4 leading to non-segregation of the YAC DNA and a consequent increase the YAC DNA copy number per cell.

5 TRANSFORMED CELLS/TRANSGENIC ORGANISMS A YAC according to the present invention may be transfered into mammalian cells by appropriately adapting the teachings of Schedl et al (reference 28). By the term "adapting" we mean following the teachings but using the vectors of the present 10 invention where appropriate. It is to be understood that other techniques may be used to transfer a YAC according to the present invention in mammalian cells and these other techniques are well documented in the art (e.g. for example see WO-A-95/14769 and/or Gietz et al 1995 Yeast vol 11 No. 4, pp 355-360).

1 5 According to Schedl et al (ibid), possibly the most straight forward approach to generate transgenic cell lines is the transfer of YACs by sphaeroblast fusion (this is a technique disclosed in the previous chapter of Reference 28). This method, however, normally leads to integration of the entire yeast genome in addition to the YAC, which might obscure the results of some experiments. In contrast, direct microinjection of DNA into 20 the nucleus of a recipient cell allows purification of a YAC prior to the transfer.

Some teachings of Schedl et al (ibid) are as follows:

Materials l. SE: 1M Sorbitol, 20mM EDTA (pH8.0).

2. TENPA: lOmM Tn's-HCI (pH 7.5), ImM EDTA (pH8.0), 100mM NaCl, 30pM spermine, 70pM spermidine.

3. IB: lOmM Tris-HCI (pH7.5), O.1mM EDTA (pH8.0), 100mM NaCl, 30pM spermine, 70pM spermidine.

41 2. Prepare a solution of 1 % Seaplaque LMP agarose in SE buffer containing 14MM P-mercaptoethanol and keep at 45 C until use.

3. Spin down cells at 4000 rpm for 5min (Sorvall RT6000). and resuspend the pellet 5 in 50m1 SE. Transfer the cell suspension into a 50m1 Falcon tube.

4. Seal the bottom of Pharmacia plug formers (insert moulds) with strips of tape and place them on ice.

1o 5. Wash cells twice with SE (4000 rpm, 5min.).

6. After the last washing step, discard the supernatant, and carefully remove all liquid by cleaning the inside of the tube with a paper towel. The cell pellet should be about 1 to 1.5m1.

7. Add 200 ul of SE buffer. With a cut off tip try to resuspend the pellet. The suspension will be very thick and difficult to pipet.

8. Transfer 0.5ml aliquots of the cell suspension into 2m1 Eppendorf tubes and keep 20 at 37 C.

9. Just before use dissolve 10mg Zymolyase in 2.5ml of the LMP agarose solution.

(Note: Zymolyase does not completely dissolve at this concentration. Weigh in the 25 required amount and work with a protein suspension.) 10. Transfer 0.5ml of this solution to the yeast cell suspension and mix thoroughly the agarose with the cells by pipetting up and down using a blue cut off tip.

30 (Note: Only a completely homogenous mixture will yield in high quality plugs with even distribution of DNA.) 43 2. Wash the high density plugs for 4x15min in TE pH8 with gentle shaking on a rocking platform.

3. Load the plugs next to one another into the preparative lane.

{Note: Best results are achieved using rectangular plugs (such as produced in Pharmacia plug formers), which can be loaded next to one another without intervening spaces. Use 90ml of the 1 % gel for a small BioRad casting chamber (14cm x 12.7cm). The plugs should occupy the entire height of the gel. Therefore, when casting the gel, make sure io that the comb is touching the bottom of the casting chamber. Make sure that the casting chamber as well as the PFGE-chamber are absolutely leveled to avoid any loss of DNA during the gel run.) Then seal the slot with 1 % LMP agarose (0.25xTAE).

4. Run the PFGE in a cooled buffer (0.25xTAE) using conditions optimized to separate the YAC from the endogenous chromosomes.

{Note: Best separation results from endogenous chromosomes are achieved using a 20 single pulse time instead of a time ramp for the entire run. It is worth while to test out several conditions before starting the isolation procedure).

5. After the gel run cut off marker lanes on either side including about 0.5cm of the preparative lane and stain them in 0.25xTAE buffer containing 0.5pg/m1 ethidium 25 bromide. Mark the position of the YAC band under UV light using a sterile scalpel blade.

6. Reassemble the gel and excise the part of the preparative lane coresponding to the YAC-DNA. Cut also slices above and below the YAC DNA to serve as marker lanes 30 for the second gel run.

Incubate for further 3h at 42 C.

15. Dialyse the resulting DNA solution for 2h on a floating dialysis membrane (Millipore, pore size 0.05gm) against microinjection buffer (IOmM Tris:HCI, pH7.5, 5 O.1mM EDTA, l00mM NaCl, 30pM spermine, 70pM spermidine).

16. To determine the DNA concentration, check 2pl on a thin 0.8% agarose gel with very small slots, using X DNA of known concentration as a standard.

io {Note: It is useful to prepare a 2ng/pl stock solution of DNA. Loading of 2, 5, 10 and 20ng of this standard should allow a relatively accurate determination of the YAC DNA concentration).

17. The integrity of the DNA can be checked running 20pl of the preparation on a 15 PFGE gel (use a comb with small slots).

Injection into cultured cells The Zeiss Automatic Injection System (AIS) can be used for rapid injection of large 2o numbers of cells growing on cell culture dishes. A digital camera attached to a microsope transmits an image to the computer screen. An interactive computer program is then used to position the pipette above a "reference cell" and to mark the tip of the needle on the screen. This position is stored by the computer and serves as a reference point for the rest of the injections. Nuclei of other cells visible on the screen can now be 25 marked by clicking on them with a computer mouse and injections are performed automatically by the computer. The amount of DNA injected can be regulated by the injection time as well as the pressure set at the Eppendorf injection system. High pressures result in higher efflux of the DNA containing solution. The pressure to be set depends on the viscosity of the DNA solution and the size of the needle opening and, 30 therefore, has to be adjusted individually in each experiment. The pressure in a standard experiment will vary between 20 and 150 hectopascal. Almost confluent dishes are best to inject. A too low cell density allows only a few cells to be injected per frame, 47 APPEND: Allows you to go back to a previous file to find the cells which have been microinjected.

NO FILE: This option does not record the cells that are injected and is sufficient for 5 most applications.

6. Select the number of frames you want to inject by filling in numbers lower than 10 for X and Y values. A frame is the window visible on the screen and, therefore, represents the field in which cells can be marked and injected at a time. Each frame has 1o specific X and Y coordinates. The computer moves along the x-axis first. An array of 5 x 10 frames will allow you to inject more than 1000 cells depending on the confluency of the plate.

7. Click on DATA OK.

8. Load 1.5p1 of DNA solution into an Eppendorf microloader and insert it into an Eppendorf Femptotip placing it at the very bottom of the tip. Slowly release the DNA solution trying to avoid the introduction of air bubbles, which can block the needle.

20 9. Twist the tip carefully to remove it from its cover and load the needle by screwing it into the injection needle holder at the microscope.

10. Choose the option adjust from the menu. Use the mouse to lower the needle by clicking onto the arrow in the center of the screen. The distance from the center 25 determines the speed of the movement. Start with high speed and slow down when you approach the surface of the dish. Once the needle touches the medium find it in low power magnification and use the micrometer screws on the pipette holder to center the needle in the frame. Change the lens to higher magnification. Focus on a plane intermediate between the cells and the needle and bring the needle down into focus. 3o Repeat this procedure until the tip of the needle is pressing down on a cell. This will result in a small halo surrounding the needle tip as you press down on the cell.

49 Too low pressure: Increase the pressure for P1. Be aware that too high pressure will result in bursting of the cells.

Press the mouse button at any time during irjections to adjust the needle height or 5 remark the tip of the needle to inject the whole frame again press RESTART. Alternatively you can carry on with CONTINUE.

13. To finish the injections press RESTART, MARKJINJECT, HOME, EXIT.

Pronuclear injections into fertilised mouse oocytes The procedure of generating transgenic mice includes isolation of fertilized oocytes from superovulated females, microinjection of DNA into pronuclei and the transfer of injected oocytes into pseudopregnant foster mothers. A detailed description of these steps can be

1 5 found in for example Hogan, Murphy and Carter (1993 Transgenesis in the mouse in "Transgenesis Techniques", Methods in Molecular Biology vol. 18 Ed. Murphy and Carter, pp 109-176. Humana Press, Totowa, New Jersey) and reference 28 (subsequent chapter) - the contents of each of which are incorporated herein by reference).

20 Preparation of DNA constructs for injection normally involves a filtration step in which the DNA is passed through a membrane with 0.2tm pore size. This step is recommended to avoid blocking of the injection needle by dust particles in the DNA solution. YAC DNA preparations should not be subjected to filtration, because of shearing forces occuring during this step. We have found that blockage of the needle is 25 a relatively infrequent event if the agarose digestion was successful. In some cases it might be preferable to centrifuge the DNA for 5min (12000rpm Eppendorf centrifuge) to remove undigested gel pieces. However, since small particles of agarose can trap DNA we would strongly recommend to determine the DNA concentration after the centrifugation step.

Some DNA preparations are very sticky, which is probably due to incomplete agarose digestion. In these cases a higher proportion of injected oocytes will be found to lyse 51 expression, regulation and function of the transgene can be analysed using simple histochemical staining procedures. This approach may provide a more complete picture of the pattern of expression of the transgene than standard procedures such as in situ hybridisation. The pYIV 1 vector can be used for YACs which have been introduced 5 into a Leu- yeast strain, while pYIV2, pYIV3, and pYIV4 can be directly introduced into the yeast strain (AB1380) which was used for construction of most YAC libraries. One of the vectors (pYIV3) permits the HA epitope tag sequence (from influenza hemagglutinin) to be fused to the carboxyl terminus of the expression product of the gene of interest, so that the protein product of the transgene can be detected using the 1o commercially available 12CA5 monoclonal antibody. pYIV4 contains IoxP elements flanking the SV40 polyadenylation signal and the ADE2 gene. In transgenic animals generated using pYIV4, the polyA sequence and the ADE2 gene sequences can be deleted using Cre recombinase so that the transgene and lacZ reporter gene are flanked by the authentic 3'-untranslated region of the transgene. A comparison between animals 15 containing the SV40 polyadenylation signal and the ADE2 gene with those in which these sequences have been removed will reveal the function of the 3' sequence of the transgene.

Expression of the IRES-IacZ from pYYV1 in YAC transgenic mice We have examined the expression of the IacZ reporter gene in transgenic mice expressing a YAC clone modified using the pYIV1 insertion vector. Insulin-like growth factor II (IGF2) cDNA was introduced 3' of the Wilm's tumor (wtl) gene promoter (isolated from a 480 kb human YAC clone). Then the promoter and cDNA were 25 inserted at the Notl-Xbal sites 5' of the IRES-IacZ-LEU2 cassette in the pYIV 1 vector. The first intron of the wtl gene was cloned 3' of the LEU2 gene. The construct was introduced to the 480 kb YAC by homologous recombination. After amplification, the modified YAC DNA was isolated and micro-injected into fertilised eggs. Eight lines of transgenic mice were produced, 5 of which expressed the lacZ reporter gene. All 30 expressing lines produced an X-Gal (it is understood that the terms X-Gal, R-Gal and LacZ are synonymous) staining pattern (Fig.5) identical to that of the human gene from 53 ICRF YAC clones 1. (49A9) 340 37 3 2. (35138) 500 103 8 5 3. (132C6) 630 133 19 Subtotal 274 30(10.9%) Chromosomal YAC clones HSC7E526 550 107 8(7.5%) E145A7 200 129 11(8.5%) ywss922 350 57 8 ywss 1545 420 59 8 ywss2056 400 15 2 ywss3844 300 151 11 Subtotal 282 29(10.3%) Total 1266 167(13.3%) The increased efficiency of retrofitting is probably due to the introduction of a 572 by Clal-Smal fragment from pYAC4 adjacent to the CEN4. When the SmaI-CIal fragment of pYAM4 was deleted by NotI-Clal digestion, or pYAM4 was linearised with Clal, the 25 frequency of tryptophan sensitive clones was not significantly different from that obtained with pCGS990.

Amplification of YAC DNA by pYAM4 Tryptophan sensitive clones were cultured in selective medium (Ura-/Lys-) with galactose as carbon source instead of glucose. In such medium, the GAL1 promoter adjacent to the CEN4 in the pYAM4 vector will be induced. Activation of transcription from the GAL1 promoter should interfere with the CEN4 leading to non-segregation of the YAC DNA therefore increase the YAC DNA copy number per cell.

As shown in Fig 7, depending on the size and nature of the insert, human YAC DNA was amplified 3 to 5 fold. Although the amplification is not as high as that achieved with pCGS990, it helps to isolate more concentrated YAC DNA for transgenics.

Lane 1 is DNA from yeast containing YAC clone 35D8; Lane 2 is DNA from yeast containing YAC clone 35D8/D6, in which the YAC has been modified by integration of the amplification vector pYAM4 and the insertion vector pYIV3; Lane 3 is the isolated YAC DNA from clone 35D8/D6 prior to microinjection. Lane 4 is DNA from yeast 5 containing YAC clone HSC7E526/V 12, in which the YAC HSC7E526 has been modified by integration of the amplification vector pYAM4 and the insertion vector pYIV3. Lane 5 is the isolated YAC DNA from clone HSC7E526/V 12 prior to microinjection.

io GENERATION OF YAC TRANSGENIC MICE Modified YAC DNA was excised from a 1 % pulse-field agarose gel in 0.25 x TAE buffer and concentrated into 4 % low melting point agarose. The gel slice containing YAC DNA was equilibrated with microinjection buffer (TE pH 7.0 with 0.1 M NaCl) 15 and digested with gelase. YAC DNA was dialysed against the microinjection buffer for 2 hours before injection into fertilised oocytes.

Two hundred and ninety-eight fertilised oocytes were injected with 35D8/D6 YAC DNA and 364 with HSC7E526/V 12. After transfer of injected oocytes into oviducts of 20 pseudopregnant female mice, a total of 190 mice (28.7%) were born of which 26 (13.7%) carried YAC DNA as determined by PCR as shown below:

Table II. Survival rate of transfered oocytes and YAC transgenic mice YAC Construct Size (kb) NEIT Born Died Transgenic 35D8/D6 500 298 97 6 18 HSC7E526/V 12 630 364 93 1 8 Total 662 190 7 26 (28.7%) (13.7%) 25 N.E. I. T: Number of oocytes injected and transferred 57 The results are presented in Figures 12 and 13. In this regard, Figure 12 shows the size of the integrated YAC DNA in transgenic founder animals carrying 35138/136 YAC DNA. Figure 13 shows the size of integrated YAC DNA in transgenic founder animals carrying HSC7E526/V12 YAC DNA. For each founder animal, the probable extent of 5 the transgene is indicated as a shaded bar, with pale circles indicating presence of markers, as determined by PCR. The location of these markers is indicated in the schematic diagram of the 35D8/D6 YAC construct (Figure 12) and HSC7E526/V 12 YAC (Figure 13) construct which are drawn above the markers.

o Three independent transgenic founder mice carrying the intact YAC 35D8/D6 (A 102.3, A102.5, A105, Figure 12) and six carrying the intact YAC HSC7E526/V 12 (A 108, A 108.1, A 108.2, A 108.3, A 108.5, A 110: Figure 13) were identified. Thus, a beneficial number of mice born in this study carried intact YAC DNA.

15 GERM LINE TRANSMISSION An important feature of pYAM4 is that it does not necessarily have to contain a thymidine kinase (TK) gene which may cause male infertility. In the absence of the TK gene, we found that YAC transgenes were transmitted into the next generation from both 20 male and female founders as determined by primer pair A, which is derived from the hygromycin resistance gene within pYAM4. Thus, preferably, the TK gene is not present in the vector of the present invention.

Immunocytochemistry for beta -galactosidase Animals were perfused transcardially with 4% paraformaldehyde in UM PBS. Brains were postfixed overnight in the same solution, then transferred into PBS next day and kept in that solution until cutting. Brains were infiltrated with 30% sucrose overnight, then 254m thick sections were cut on a cryostat. Sections were collected in multiwell plates 30 containing PBS and then processed for immunocytochemistry. Sections were treated with 0.1% Triton X-100 and 0.02% HZO2 solution for 30 min, then rinsed two times for 5 min with PBS. This was followed by a blocking step with 2% solution of normal donkey 59 acting through the VIP2 receptor, may play a role in the control of circadian rhythms. There is circadian variation of VIP immunoreactivity (Takahashi et al., 1989), prepro VIP mRNA (Albers et al., 1990; Glazer and Gozes, 1994; Gozes et al., 1989) and of VIP2 receptor mRNA (Cagampang et al., 1998) in the SCN and VIP (Piggins et al., 5 1995), VIP antagonists (Gozes et al., 1995) and VIP antisense oligodeoxynucleotides (Scarbrough et al., 1996) have been shown to disrupt circadian function.

(ii) In the pancreas (Figure 5c), where VIP and PACAP stimulate insulin release by interaction with the VIP2 receptor on the beta cell (Straub and Sharp, 1996).

Chemiluminescent assay for P-galactosidase in mouse tissues using the Tropix Galacto-Light Plus kit.

Tissues from mice were dissected, frozen on dry ice and stored at -700C. They were 15 thawed and homogenised immediately in 100-4001i1 of cold lysis buffer (as supplied in the kit, with 0.2mM PMSF and 514g/ml leupeptin added just before use). After homogenisation, samples were centrifuged at 12000g for 10 min at 4 C. An aliquot of the supernatant was stored at -70 C for measurement of protein concentration and the rest was incubated at 48 C for 60 minutes to inactivate the endogenous pgalactosidase. 20 After centrifugation for 5 min at room temperature 201. 1 of each sample were used in the assay. 2001l of Galacto-Light reaction buffer was added, inbubated for 60 min at room temperature and then 30011 of Accelerator was added and the sample counted in a TD 20/20 luminometer (for 20 sec after 5 sec delay). Protein concentrations were determined using the Bio- Rad assay and activity was expressed as light units/min/mg of 25 protein.

61 between two YAC vector arms, facilitating transfer of a whole gene and most (if not all) of the elements required for its faithful regulation into transgenic animals.

The analysis in transgenic animals of genes expressed in YACs can be greatly facilitated 5 by the use of a reporter gene in accordance with the present invention for the accurate and sensitive detection of cellular sites of transcription.

AMPLIFICATION VECTOR 10 Importance of YAC technology and YAC transgenesis The development of the yeast artificial chromosome (YAC) technology has permitted the cloning of DNA segments thousands of kilobases in size between two YAC vector arms. YACs can be used to clone the complete sequences of large genes or gene complexes 15 that exceed the size limit for cloning in conventional bacterial cloning vectors such as plasmids (10 kb), bacteriophage (15 kb), and cosmids (50 kb). Cloning of such large DNA fragments is essential for physical genome mapping (1) and to isolate large genes relevant to human genetic disease (2, 3). Although bacterial artificial chromosome (BAC) (4) and P1 artificial chromosome vectors (5) have a large cloning capacity (up to 2o 200 kb), it is relatively difficult to perform genetic manipulation in these vectors. The high efficiency of homologous recombination in yeast permits genetic manipulations of genes cloned in YAC vectors to be performed easily.

Transgenic technology has played an important role in the understanding of gene 25 function and regulation in vivo, and in creating animal models of human genetic diseases. It is well recognised that transgenes containing genomic DNA with introns and essential regulatory sequences are expressed more appropriately in vivo than cDNA based constructs (6- 10). The use of YAC constructs to produce transgenic animals facilitates the presence and transfer of most (if not all) elements required for the faithful regulation 30 of a gene and may avoid position effects related to the integration site, which may lead to low levels and, in some cases, aberrant patterns of gene expression in transgenic 63 heterologous (herpes simplex virus) thymidine kinase (TK) gene. YAC DNA of less than 600 kb is amplified efficiently (3 to 11 copies/cell). pCGS966 has been used recently to construct a number of new YAC libraries (13-15). However, when using this vector for the modification (retrofitting) of existing YACs, replacement of the left arm occurs with 5 very low frequency (0.5-2.5 %) (12). Most importantly, the expression of the TK gene in the testes of transgenic mice interferes with spermatogenesis and causes male infertility (16-22). This complication makes these vectors unsuitable for transgenic studies.

Features of the novel YAC amplification vector pYAM4.

We have constructed a YAC amplification vector which has a number of advantages:

1) it amplifies YAC DNA 3 to 5 fold.

15 2) unlike existing vectors, it does not contain the herpes simplex virus thymidine kinase (TK) gene, which causes male infertility in transgenic mice.

3) it has much higher homologous recombination efficiency (13%) than existing YAC amplification vectors.

4) it contains a selectable marker (hygromycin B resistance) which facilitates the transfer of YAC DNA into embryonic stem cells and other cell lines.

5) it can be used for targeted deletion of sequences cloned in YAC vectors.

6) Fusion of a NOI (such as the SERT gene) to a reporter gene (such as LacZ) facilitates the determination of the sites/regions where the NOI is expressed and the testing of agents which may affect the expressionpattern of the NOI.

30 7) Transgenic mice overexpressing the human VIPR2 gene together with the (3galactosidase reporter gene will facilitate the development of agents capable of influencing the activity of the VIP2 receptor in man. Two classes of agent might be transgenes. These vectors are likely to find widespread application in transgenic research.

In these studies we also present the construction of a YAC amplification vector (such as 5 pYAM4) which has a number of advantages over previous vectors and is suitable for the amplification of YAC DNA for the creation of transgenic mice.

In these studies we also present the combined use of the above-mentioned modification vectors (such as pYIVl, pYIV2, pYIV3 and pYIV4) and the above-mentioned YAC 1o amplification vector (such as pYAM4). The combined use of these vectors is likely to find widespread application in transgenic research.

For example, the vectors of the present invention - in particular the insertion vectors of the present invention - may be used to prepare other artificial chromosomes (i.e. 15 artificial chromosomes other than YACs), which may in turn be used to prepare transgenic organisms (including animals). In this alternative embodiment, the above mentioned statements of invention and description are still applicable but wherein the term YAC represents any suitable artificial chromosome, preferably a yeast artificial chromosome.

All publications mentioned in the above specification are herein incorporated 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 25 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.

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71 721 TGGGACAGTA CCACCGAAAT GGATGCATTT CAATATGGAG GAAAATCTGC CCGATTTTCA 781 AAGGGATTGG TTATGCCATC TGCATCATTG CCTTTTACAT TGCTTCCTAC TACAACACCA 841 TCATGGCCTG GGCGCTATAC TACCTCATCT CCTCCTTCAC GGACCAGCTG CCCTGGACCA 901 GCTGCAAGAA CTCCTGGAAC ACTGGCAACT GCACCAATTA CTTCTCCGAG GACAACATCA 5 961 CCTGGACCCT CCATTCCACG TCCCCTGCTG AAGAATTTTA CACGCGCCAC GTCCTGCAGA 1021 TCCACCGGTC TAAGGGGCTC CAGGACCTGG GGGGCATCAG CTGGCAGCTG GCCCTCTGCA 1081 TCATGCTGAT CTTCACTGTT ATCTACTTCA GCATCTGGAA AGGCGTCAAG ACCTCTGGCA 1141 AGGTGGTGTG GGTGACAGCC ACCTTCCCTT ATATCATCCT TTCTGTCCTG CTGGTGAGGG 1201 GTGCCACCCT CCCTGGAGCC TGGAGGGGTG TTCTCTTCTA CTTGAAACCC AATTGGCAGA 10 1261 AACTCCTGGA GACAGGGGTG TGGATAGATG CAGCCGCTCA GATCTTCTTC TCTCTTGGTC 1321 CGGGCTTTGG GGTCCTGCTG GCTTTTGCTA GCTACAACAA GTTCAACAAC AACTGCTACC 1381 AAGATGCCCT GGTGACCAGC GTGGTGAACT GCATGACGAG CTTCGTTTCG GGATTTGTCA 1441 TCTTCACAGT GCTCGGTTAC ATGGCTGAGA TGAGGAATGA AGATGTGTCT GAGGTGGCCA 1501 AAGACGCAGG TCCCAGCCTC CTCTTCATCA CGTATGCAGA AGCGATAGCC AACATGCCAG 15 1561 CGTCCACTTT CTTTGCCATC ATCTTCTTTC TGATGTTAAT CACGCTGGGC TTGGACAGCA 1621 CGTTTGCAGG CTTGGAGGGG GTGATCACGG CTGTGCTGGA TGAGTTCCCA CACGTCTGGG 1681 CCAAGCGCCG GGAGCGGTTC GTGCTCGCCG TGGTCATCAC CTGCTTCTTT GGATCCCTGG 1741 TCACCCTGAC TTTTGGAGGG GCCTACGTGG TGAAGCTGCT GGAGGAGTAT GCCACGGGGC 1801 CCGCAGTGCT CACTGTCGCG CTGATCGAAG CAGTCGCTGT GTCTTGGTTC TATGGCATCA 20 1861 CTCAGTTCTG CAGGGACGTG AAGGAAATGC TCGGCTTCAG CCCGGGGTGG TTCTGGAGGA 1921 TCTGCTGGGT GGCCATCAGC CCTCTGTTTC TCCTGTTCAT CATTTGCAGT TTTCTGATGA 1981 GCCCGCCACA ACTACGACTT TTCCAATATA ATTATCCTTA CTGGAGTATC ATCTTGGGTT 2041 ACTGCATAGG AACCTCATCT TTCATTTGCA TCCCCACATA TATAGCTTAT CGGTTGATCA 2101 TCACTCCAGG GACATTTAAA GAGCGTATTA TTAAAAGTAT TACCCCAGAA ACACCAACAG 25 2161 AAATTCCTTG TGGGGACATC CGCTTGAATG CTGTGTAACA CACTCACCGA GAGGAAAAAG 2221 GCTTCTCCAC AACCTCCTCC TCCAGTTCTG ATGAGGCACG CCTGCCTTCT CCCCTCCAAG 2281 TGAATGAGTT TCCAGCTAAG CCTGATGATG GAAGGGCCTT CTCCACAGGG ACACAGTCTG 2341 GTGCCCAGAC TCAAGGCCTC CAGCCACTTA TTTCCATGGA TTCCCCTGGA CATATTCCCA 2401 TGGTAGACTG TGACACAGCT GAGCTGGCCT ATTTTGGACG TGTGAGGATG TGGATGGAGG 30 2461 TGATGAAAAC CACCCTATCA TCAGTTAGGA TTAGGTTTAG AATCAAGTCT GTGAAAGTCT 2521 CCTGTATCAT TTCTTGGTAT GATCATTGGT ATCTGATATC TGTTTGCTTC TAAAGGTTTC 2581 ACTGTTCATG AATACGTAAA CTGCGTAGGA GAGAACAGGG ATGCTATCTC GCTAGCCATA 2641 TATTTTCTGA GTAGCATATA GAATTTTATT GCTGGAATCT ACTAGAACCT TCTAATCCAT 2701 GTGCTGCTGT GGCATCAGGA AAGGAAGATG TAAGAAGCTA AAATGAAAAA TAGTGTGTCC 35 2761 ATGCAAGCTT GTGAGTCTGT GTATATTGTT GTTTCAGTGT ATTCTTATCT CTAGTCCAAT 2821 ATTTTGGGCC CATTACAAAT ATATGAATTC CCCAAATTTT TCTTACATTA ACAAATTCTA 2881 CCAACTCAA 40 SEQ ID. NO. 3 LOCUS hVIP2.DOC 3944 BP DS-DNA ENTERED 11/23/98 BASE COUNT 795 A 1159 C 1154 G 836 T 0 OTHER 45 COMMENT 1 - 238 exon 1 239 - 338 exon 2 339 - 446 exon 3 447 - 544 exon 4 545 - 642 exon 5 50 643 784 exon 6 785 - 935 exon 7 936 - 996 exon 8 997 - 1066 exon 9 1067 - 1158 exon 10 55 1159 - 1288 exon 11 1289 - 1330 exon 12 1331 - 3944 exon 13 188 - 1504 open reading frame encoding VIP2 receptor ORIGIN - 60 1 GTGCATTGAG CGCGCTCCAG CTGCCGGGAC GGAGGGGGCG GCCCCCGCGC TCGGGGCGCT 61 CGGCTACAGC TGCGGGGCCC GAGGTCTCCG CGCACTCGCT CCCGGCCCAT GCTGGAGGCG 121 GCGGAACCGC GGGGACCTAG GACGGAGGCG GCGGGCGCTG GGCGGCCCCC GGCACGCTGA 181 GCTCGGGATG CGGACGCTGC TGCCTCCCGC GCTGCTGACC TGCTGGCTGC TCGCCCCCGT 73 SEQ ID NO. 4 GAATTCCGCC CCTCTCCCTC CCCCCCCCCT AACGTTACTG GCCGAAGCCG CTTGGAATAA GGCCGGTGTG CGTTTGTCTA TATGTTATTT TCCACCATAT 5 TGCCGTCTTT TGGCAATGTG AGGGCCCGGA AACCTGGCCC TGTCTTCTTG ACGAGCATTC CTAGGGGTCT TTCCCCTCTC GCCAAAGGAA TGCAAGGTCT GTTGAATGTC GTGAAGGAAG CAGTTCCTCT GGAAGCTTCT TGAAGACAAA CAACGTCTGT AGCGACCCTT TGCAGGCAGC GGAACCCCCC ACCTGGCGAC AGGTGCCTCT GCGGCCAAAA GCCACGTGTA TAAGATACAC CTGCAAAGGC 10 GGCACAACCC CAGTGCCACG TTGTGAGTTG GATAGTTGTG GAAAGAGTCA AATGGCTCTC CTCAAGCGTA TTCAACAAGG GGCTGAAGGA TGCCCAGAAG GTACCCCATT GTATGGGATC TGATCTGGGG CCTCGGTGCA CATGCTTTAC ATGTGTTTAG TCGAGGTTAA AAAACGTCTA GGCCCCCCGA ACCACGGGGA CGTGGTTTTC CTTTGAAAAA CACGATGATA AGCTTGCCAC AACCATG (this sequence is also presented as Figure 10).

12. A vector capable of modifying a YAC or a YAC vector wherein the vector comprises a nucleotide sequence wherein the nucleotide sequence comprises the sequence presented as SEQ ID No. I or SEQ ID No. 4 or a variant, homologue or derivative thereof.

13. pYAM4.

14. A YAC prepared by one or more of the vectors according to any one of the preceding claims.

15. Use of an IRES to modify a YAC vector or a YAC.

16. Use of a nucleotide sequence comprising the sequence presented SEQ ID No. I or SEQ ID No. 4 or a variant, homologue or derivative thereof in a vector to prepare a 15 YAC vector or a YAC.

17. Use of a nucleotide sequence comprising the sequence presented as SEQ ID No. 1 or SEQ ID No. 4 or a variant, homologue or derivative thereof in a vector to increase the expression efficiency of one or more NOIs within a YAC vector or a YAC.

18. A YAC transgenic mammal co-expressing an NOI and a reporter gene wherein the expression pattern of the NOI can be determined by measuring a detectable signal (such as a visually or an immunologically detectable signal) produced by the expression product of the reporter gene.

19. A YAC transgenic mammal expressing a reporter gene under the control of a regulatory sequence from a human NOI.

20. Use of a YAC transgenic mammal to test for potential pharmaceutical and/or 30 veterinary agents.

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

26. A process comprising the steps of:

(a) performing the assay according to claim 21 or claim 22; (b) identifying one or more agents that affect the expression pattern of the NOI or the EP activity thereof; to (c) modifying one or more identified agents to cause a different effect on the expression pattern of the NOI or the EP activity thereof.

27. Use of a YAC according to claim 2 or claim 3 or claim 4 or claim 5 or any one 15 of the vectors described in claims 6 to 9 or claim 13 (including combinations thereof) to screen for agents capable of affecting the expression pattern of an NOI or the EP activity thereof in a transgenic mammal.

28. Use of an agent in the preparation of a pharmaceutical composition for the 20 treatment of a disorder or condition associated with the expression pattern of an NOI or the EP activity thereof, the agent having an effect on the expression pattern of the NOI or the EP activity thereof when assayed in vitro by the assay method according to claim 21 or claim 22.

25 29. Use of an agent identified by an assay according to claim 21 or claim 22 in the manufacture of a medicament which affects the expression pattern of an NOI or the EP activity thereof.

30. Use of an agent in the preparation of a pharmaceutical composition for the 30 treatment of a disorder or condition associated with the expression pattern of an NOI or the EP activity thereof, the agent having an effect on the expression pattern of the NOI