WO1999028449A2 - Yac vectors - Google Patents
Yac vectors Download PDFInfo
- Publication number
- WO1999028449A2 WO1999028449A2 PCT/GB1998/003558 GB9803558W WO9928449A2 WO 1999028449 A2 WO1999028449 A2 WO 1999028449A2 GB 9803558 W GB9803558 W GB 9803558W WO 9928449 A2 WO9928449 A2 WO 9928449A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- yac
- vector
- noi
- dna
- activity
- Prior art date
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
Definitions
- the present invention relates to vectors, in particular vectors that are suitable for use as 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.
- it is generally possible to clone into a YAC stretches of DNA that are up to about lOOOkb 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 ⁇ (typically up to 25kb stretches of DNA), cosmids (typically up to 45kb stretches of DNA) and the PI vector (typically up to lOOkb stretches of DNA) (see Lodish et al 1995 Molecular Cell Biology 3rd 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 have been presented by T.A. Brown 1995 (Gene Cloning, An Introduction, 3rd Edition, page 325, Pub. Chapman & Hall, pages 139-142).
- a YAC typically contains the following essential functional elements: a centromere, two telomeres and one or more origins of replication.
- the centromere is required to 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.
- YACs are typically prepared from YAC vectors. These vectors are typically circular. When they are needed to be used to prepare the YAC they are then linearised - such as by use of specific restriction enzymes.
- YAC vectors have been proposed in the literature. Examples of such vectors are pYAC2 and pYAC4 which are discussed in US-A-4889806. Other YAC vectors are disclosed in WO-A-95/03400. Other YAC vectors include pYAC3 and pYAC5.
- YAC vectors - such as pYAC3, pYAC4 and pYAC5 - are essentially a pBR322 plasmid into which a number of yeast genes have been inserted. These genes include a yeast centromere region (called CEN4), two telomere regions (called TEL), and two selectable marker genes (called URA3 and TRP1).
- CEN4 yeast centromere region
- TEL telomere regions
- URA3 and TRP1 selectable marker genes
- the TEL sequences do not correspond to the full genomic telomere sequences. Nevertheless these partial sequences still function as telomeric sequences.
- One replication origin (called ori, such as ARSl) is positioned intermediate CEN4 and TRP1.
- the YAC vectors When used for cloning, the YAC vectors are cut with BamHl and a second restriction enzyme (Sw l for pYAC3, EcoRI for pYAC4 and Notl for pYAC5) to produce two vector arms. The fragments are then ligated with a nucleotide sequence of interest (which for ease of reference shall be called "NOI”) which has been digested with corresponding restriction enzymes.
- a second restriction enzyme Sw l for pYAC3, EcoRI for pYAC4 and Notl for pYAC5
- This resultant linear structure (which is not drawn to scale) is presented as Figure 17.
- This resultant linear structure - which is a YAC - comprises in the correct orientation the essential functional features of a chromosome.
- YACs have been - and are still being - used to map the human genome.
- NOI is a gene or fragment thereof whose full sequence (or even function) may not have been determined.
- YACs are used to create genomic libraries which are then screened.
- the physical map of the human Y chromosome and the long arm of chromosome 21 have been determined through analysis of long segments of human DNA cloned into YACs by ter alia sequence tagged sites. This work is summarised in Lodish et al 1995 (ibid, page 285).
- the use of YACs is by no means limited to mapping of the human genome.
- YACs have 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 the Arabidopsis genome.
- YACs 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 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 255-258). By way of further example, WO-A-94/23049 reports on a YAC containing the gene coding for ⁇ -amyloid precursor protein.
- 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 function of the NOI need not be necessary.
- YACs may even be used to study the functional behaviour of mutant genes.
- WO-A-95/14769 reports on a method of producing a mouse that expresses human mutant protein sequences that utilises the "pop-in/pop-out" method in combination with YAC technology to insert mutations into YACs and thereby derive stem cells capable of being used in the development of transgenic mice.
- This particular method comprises obtaining a gene contained within a YAC, introducing a predetermined mutant human DNA sequence (which is the NOI) into the YAC by homologous recombination, utilising transgenics to insert the mutant gene into embryonic stem cells, and injecting the stem cells into blastocysts to derive a transgenic mouse that expresses the mutant protein sequences.
- a predetermined mutant human DNA sequence which is the NOI
- the "pop-in/pop-out” method is described by Rothstein (1991 Methods In Enzymology vol 194, Guide To Yeast Genetics and also in Molecular Biology. Eds. Gutherie at al, San Diego: Academic Press. Pages 281-301) and McCormick et al (TCM Vol 6 No. 1 1996 pages 16-24).
- YACs have many important applications, there are nevertheless problems associated with their production and their usage. 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 pi of 500kb YAC at a concentration of 1 ng/ ⁇ l is injected into a 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.
- YAC vector pCGS990 Smith et al Mammalian Genome 1993 4 pages 141-147.
- TK gene i.e. the herpes simplex virus thymidine kinase gene
- the present invention seeks to improve upon the existing techniques associated with the preparation of and usage of YACs.
- 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 first aspect of the present invention there is provided one or more of the following embodiments, which for ease have been presented as numbered paragraphs:
- a YAC vector comprising an IRES.
- a YAC vector comprising a reporter gene wherein the expression product of the reporter gene is capable of producing a visually detectable signal.
- a YAC vector comprising a reporter gene wherein the expression product of the reporter gene is capable of producing an immunologically detectable signal.
- a YAC vector comprising an IRES and a reporter gene wherein the expression product of the reporter gene is capable of producing a visually detectable signal.
- a YAC vector comprising an IRES and a reporter gene wherein the expression product of the reporter gene is capable of producing an immunologically detectable signal.
- a YAC vector comprising a nucleotide sequence wherein the nucleotide sequence comprises the sequence presented as SEQ ID No.l or SEQ ID No. 4 a variant, homologue or derivative thereof.
- a YAC prepared by the vector according to any one of the above-mentioned embodiments.
- nucleotide sequence comprising the sequence presented SEQ ID No.l or SEQ ID No. 4 or a variant, homologue or derivative thereof in a vector to prepare a nucleotide sequence comprising the sequence presented SEQ ID No.l or SEQ ID No. 4 or a variant, homologue or derivative thereof in a vector to prepare a nucleotide sequence comprising the sequence presented SEQ ID No.l or SEQ ID No. 4 or a variant, homologue or derivative thereof in a vector to prepare a
- YAC vector or a YAC.
- nucleotide sequence comprising the sequence presented SEQ ID No.l 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.
- 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.
- the term "vector" as used herein in the general sense means the vector of the first aspect of the present invention and/or the vector of the second aspect of the present invention.
- a third aspect of the present invention there is provided the combination of at least any one of the embodiments of the first aspect of the present invention and at least any one of the embodiments of the second aspect of the present invention.
- the term "combination" means that the resultant YAC or transgenic/transformed cell, organ or organism is prepared by use of both the vector of the first aspect of the present invention and the vector of the second aspect of the present invention.
- the third aspect of the present invention is not limited to preparative techniques wherein both the vector of the first aspect of the present invention and the vector of the second aspect of the present invention have to be used at the same time when preparing the YAC, let alone the transformed/transgenic cell, organ or organism.
- a YAC vector or a YAC comprising a selection gene, wherein that selection gene is specifically removable from the YAC vector or the YAC.
- 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 yy umw PCT/GB98/03558
- a YAC transgenic mammal expressing a reporter gene under the control of a regulatory sequence from a human NOI.
- YAC transgenic mammal Use of a YAC transgenic mammal to test for potential pharmaceutical and/or veterinary agents.
- 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 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.
- a process comprising the steps of:
- a process comprising the steps of:
- a YAC according to the present invention or any one of the vectors according to the present invention to screen for agents capable of affecting the expression pattern of an NOI or the EP activity thereof in a transgenic mammal.
- an agent in the preparation of a pharmaceutical composition for the 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 according to the present invention.
- At least part of the assay can be carried out in living tissue.
- 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.
- SERT human serotonin transporter
- SERT human serotonin transporter
- variant 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. 2, preferably having at least the same level of activity of the expression product of SEQ I.D. No. 2.
- homologue covers 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.
- VIP2 receptor VIPR2
- VIP2 receptor is referred to interchangeably throughout the text as the VIP2 receptor, VIPR2 or VPAC2R (Harmar et al 1998 Pharmacological Reviews 50: 265- 270).
- the VIPR2 is the VIP 2 receptor (VIPR2) presented as SEQ ID No. 3 or a variant, homologue or derivative thereof.
- variant 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. 3, preferably having at least the same level of activity of the expression product of SEQ I.D. No. 3.
- homologue covers 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.
- the present invention also encompasses modified YACs 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.
- affect includes any one or more of: treats, prevents, suppresses, alleviates, restores, modulates, influences or to otherwise alter an existing state.
- agent includes any entity (such as one or more chemical compounds, including peptide sequunces and variants/homologues/derivatives/fragments thereof) which is capable of affecting the expression pattern of the NOI or the EP activity 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.
- EP expression product
- expression product 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.
- disurbances in circadian function means disorders which may lead to impaird physical and mental well-being that can occur through extremes in work patterns such as shift work, in normal ageing, when travelling through time zones (jet lag), and in dementia.
- the insertion vector it is possible to readily monitor the in vivo expression pattern of a NOI.
- the insertion vector it is possible to express - such as over-express - a NOI and a reporter gene contained within or as a YAC.
- the vector may comprise at least one NOI.
- the term NOI i.e. nucleotide sequence of interest
- 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.
- the DNA sequence can be in a sense orientation or in an anti-sense orientation. Preferably, it is in a sense orientation.
- the DNA is or comprises cDNA.
- the vectors of the present invention may be used to prepare a modified YAC or a modified YAC vector.
- the modified YAC or the modified YAC vector can be used, for example, for expression and/or regulation and/or functional studies of the NOI.
- the vectors of the present invention can be used to prepare modified YACs or modified YAC vectors that can be used for expression and/or regulation and/or functional studies of the NOI in combination with other entities, such as other NOIs, compounds or compositions.
- the modified YAC or modified YAC vector can be used to test potential pharmaceutical agents (including veterinary agents).
- modified YAC or modified YAC vector means a modified YAC or modified YAC vector having a modified genetic structure.
- the YAC or YAC vector has a modified genetic structure since part or all of the vector according to the present invention has been incorporated into the YAC or YAC vector.
- the vectors of the present invention may be used to prepare transformed cells that can be used, for example, for functional studies of the NOI.
- the vectors of the present invention can be used to prepare transformed cells that can be used for functional studies of the NOI in combination with other entities, such as other NOIs, compounds or compositions.
- the transformed cells can be used to test potential pharmaceutical agents (including veterinary agents).
- 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).
- transformed cell means a cell having a modified genetic structure.
- the cell has a modified genetic structure since a vector according to the present invention has been introduced into the cell.
- cell includes any suitable organism.
- the cell is a mammalian cell.
- 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 or in vitro, or combinations thereof.
- the cell will be transformed by any one of the following methods: transfection, microinjection, electroporation or microprojectile bombardment, including combinations thereof.
- the cell will be transformed by, or by at least, transfection.
- 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.
- 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, compounds or compositions.
- the transgenic organisms can be used to test potential pharmaceutical agents (including veterinary agents).
- the term "transgenic organism” means an organism comprising a modified genetic 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.
- organism includes any suitable organism.
- the organism is a mammal.
- the organism is a mouse.
- the transgenic organisms may be prepared by use of the transformed cells of the present invention.
- the insertion vector of the present invention is itself a YAC vector.
- the amplification vector of the present invention is itself a YAC vector.
- the vector of the present invention may additionally comprise one or more selection genes to enable the vector and any resultant entity comprising the same or made from the same (such as a modified YAC vector, a YAC or a specific yeast strain comprising any one of the same) to be selectively grown and/or screened.
- selection genes can be chosen from suitable selection genes that are available. Examples of suitable selection genes include LYS2 (see Barnes and Thorner 1986, Mol and Cell Biol 6: pp 2828-2838), LEU2 (see Beach and Nurse 1981 Nature vol 290 pp 140-142), d ADE2 (see Stotz and Linder 1990 Gene 95 pp 91-98).
- any one or more of the selection gene is specifically removable from the vector, the modified YAC vector and the modified YAC according to the present invention.
- the term "specifically removable” means being able to remove the one or more selection gene without disrupting any other region in the vector, the modified YAC vector and the modified YAC according to the present invention.
- the selection gene may be flanked by unique restriction sites.
- the selection gene may be flanked by a LoxP element which is removable by use of Cre recombinase.
- 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 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 present on the YAC.
- the YAC vector may additionally comprise one or more NOIs.
- the NOI need not be of known function and/or structure.
- the NOI is of human origin.
- the YAC vector comprises an internal ribosomal entry site (i.e. an IRES).
- IRES internal ribosomal entry site
- 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 YAC.
- IRES sequences act on improving translation efficiency of RNAs in contrast to a promoter's effect on the transcription of DNAs.
- IRES sequences include those from encephalomyocarditis virus (EMCV) (Ghattas, I.R., et al., Mol. Cell.
- 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).
- the present invention is not intended to be limited to a specific IRES sequence.
- 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. 1 or a variant, homologue, derivative or fragment thereof.
- variant 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. 1.
- homologue covers 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.
- IRES sequence is that presented as SEQ ID No. 4 or a variant, homologue, derivative or fragment thereof.
- variant 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.
- homologue covers 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.
- 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).
- 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.
- 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 incorporated herein by reference.
- the search parameters are defined as follows, can be advantageously set to the defined default parameters.
- substantially identical 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.
- BLAST Basic Local Alignment Search Tool
- 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 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.
- blastp - compares an amino acid query sequence against a protein sequence database.
- 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).
- 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.
- 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.
- 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.
- NCBI-gi causes NCBI gi identifiers to be shown in the output, in addition to the accession and/or locus name.
- sequence comparisons are conducted using the simple BLAST search algorithm provided at http://www.ncbi.nlm.nih.gov/BLAST.
- 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.
- 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).
- 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.
- 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.
- 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.
- stringent conditions eg. 65°C and O.lxSSC ⁇ lxS
- Insertion of the IRES into a YAC by use of the insertion vector of the present invention - and thus forming a modified YAC - enables the modified YAC to express, in particular over-express, at least two nucleotide sequences.
- these two nucleotide sequences one may be a NOI, such as a NOI of human origin.
- the other of those nucleotide sequences may be another NOI.
- the other nucleotide sequence is a reporter gene according to the present invention.
- the present invention also encompasses a vector or a YAC obtained therefrom comprising more than one IRES.
- the vector or the YAC obtained therefrom preferably comprises more than NOI.
- the YAC vector comprises a reporter gene whose expression product is capable of producing a visually detectable signal.
- 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 Frevier 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).
- 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.
- 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).
- 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
- FLAG epitope Formd et al 1991 Protein Expr Purif Apr; 2(2):95-107.
- a reporter gene 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.
- 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 ability or pattern of that regulatory element.
- 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 nucleotide sequences) the NOI to the reporter gene according to the present invention.
- workers can readily determine which sites or regions that NOI is expressed.
- 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.
- One aspect of the present invention concerns the use of SEQ ID No. l or SEQ ID No. 4 or a variant, homologue or derivative thereof.
- variant, homologue or derivative thereof includes any addition, substitution or deletion of one or more nucleic acids providing the resultant entity can still function as an IRES.
- any variant, homologue or derivative of the IRES sequence comprises at least 100 bp of SEQ ID No.l or SEQ ID No. 4.
- any variant, homologue or derivative comprises at least 200 bp of SEQ ID No.l or SEQ ID No. 4.
- any variant, homologue or derivative comprises at least 300 bp SEQ ID No.l or SEQ ID No. 4.
- any variant, homologue or derivative comprises at least 400 bp of SEQ ID No. l or SEQ ID No. 4.
- any variant, homologue or derivative comprises at least 500 bp of SEQ ID No.l or SEQ ID No. 4.
- sequence identity there is at least 80% sequence identity, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferably there is at least 98% sequence identity with the sequence shown as SEQ ID No.l or SEQ ID No. 4.
- nucleotide sequence is the sequence presented as SEQ ID No.l or SEQ ID No. 4.
- the YAC vector of the present invention comprises the nucleotide sequence presented as SEQ ID No. l or SEQ ID No. 4 or a variant, homologue or derivative thereof.
- the nucleotide sequence increases the expression efficiency of one or more NOIs within a YAC vector or a YAC.
- 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).
- 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 above-mentioned aspects.
- JM109 pYIVl deposit number NCIMB 40907
- JM109 pYIV2 deposit number NCIMB 40908
- JM109 pYIV3 deposit number NCIMB 40909
- JM109 pYIV4 deposit number NCIMB 40910 JM109 pYAM4 - deposit number NCIMB 40906
- Figure 1 which is a diagrammatic representation of pYIVl
- FIG. 2 which is a diagrammatic representation of pYIV2
- FIG. 3 which is a diagrammatic representation of pYIV3 ;
- FIG 4 which is a diagrammatic representation of pYIV4;
- Figure 5 which is a photographic image;
- FIG. 6 which is a diagrammatic representation of pYAM4
- Figure 7 which is a picture of a gel
- Figure 8 which is a photographic image
- Figure 11 which is a picture of a gel
- Figure 12 which is a PCR map of the integrated SERT 35D8/D6 YAC DNA
- Figure 13 which is a PCR map of the integrated VIPR2 HSC7E526/V12 YAC DNA
- Figure 14 which is a photographic image
- Figure 15 which is a photographic image
- Figure 16 which is a graphical representation of ⁇ -Gal enzyme activity determined using a chemiluminescent reporter assay system
- FIG. 17 which is a schematic diagram.
- Figure 7 shows amplication of YAC DNA by pYAM4.
- the endogenous chromosomal DNA from S. cerevisiae is shown in lane 3 and sizes in kb at the left. All other lanes were loaded with DNA plugs form Lys+ YAC clones and cultured in medium with galactose but lacking lysine after retrofitting with pYAM4.
- the migration position of YAC DNA in each clone is indicated with an arrow.
- Levels of amplification of the YAC DNA are based on comparison of ethidium bromide staining of YAC DNA to that of endogeous chromosomes of similar size.
- Lane 1 350 kb, 8-fold, lane 2, 630 kb, 3-fold, lane 4, 615 kb, 3-fold, lane 5, 500 kb, 4-fold, lane 6, 200 kb, 5-fold, lane 7, 200 kb, 6-fold; lane 8, 230 kb, 2-fold; lane 9, 150 kb, 3-fold, lane 10/11/12, 230 kb, 5-fold.
- Figure 10 shows the IRES sequence which is derived from Encephalomycocarditis virus. The sequence has been genetically modified at the 3' end to introduce a Hindlll restriction site.
- 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 ⁇ -galactosidase activity in transgenic mice containing the YAC HSC7E526/V12.
- Figures 15a and 15b show staining patterns for ⁇ -galactosidase activity in a coronal slice from the brain of a transgenic mouse for whom mouse A108.2 was the father.
- the stained suprachiasmatic nuclei are indicated with arrowheads
- 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.
- Figure 16 illustrates the tissue distribution of ⁇ -galactosidase activity in transgenic mice containing the YAC HSC7E526/V12.
- ⁇ -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 ⁇ -galactosidase activity was detected.
- the vector can be used for YACs which have been introduced into a Leu " yeast strain. The details of the construction procedure were as follows:
- a 3.5 kb cassette containing the lacZ gene and polyadenylation sequences was isolated from pMR ⁇ -/ cZ-PA (23) by Sail (complete) and EcoRI (partial) digestion and inserted into the EcoRI-S ⁇ /I sites of pBluescript SK " , generating pSK ⁇ / ⁇ cZ-PA.
- the IRES was introduced into pSK ⁇ / ⁇ cZ-PA by replacing the 1.1 kb Xb ⁇ l (in the polylinker) -EcoRV (in the lacZ sequence) fragment with a 1.7 kb Xbal-EcoRV fragment (IR ⁇ S-5 '-lacZ) from pIRES-bgeo (24), resulting in pIRES-Z ⁇ cZ-PA.
- pYIV2 is similar to pYIVl except that the LEU2 gene is replaced by the ADE2 gene so that the plasmid can be directly introduced into the conventional yeast strain (AB1380) used for the construction of most YAC libraries.
- a 2.5 kb Bg ⁇ l fragment containing the ADE2 gene was isolated from pASZll, filled in and inserted into the filled in Hindlll 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-/ ⁇ cZ-PA cassette was released from pIRES-/ ⁇ cZ-PA by Sail digestion, filled in and then cut with Notl.
- the fragment was inserted into the EcoRI (filled in) and Notl sites of pSK"AD ⁇ 2, resulting a plasmid (pYIV2) containing and IRES-/ ⁇ cZ-ADE2 cassette (Fig.4).
- pYIV2 plasmid containing and IRES-/ ⁇ cZ-ADE2 cassette
- HA haemaggluttinin
- the translation stop codon of the human VIP2 receptor cloned in the vector pcD ⁇ A3 was converted into an Xbol site by PCR-based mutagenesis.
- a linker encoding the HA epitope tag flanked by Xhol-Xb ⁇ l sites was converted into an Xbol site by PCR-based mutagenesis.
- the VIP2 receptor- HA fragment was released from the pcDNA3 vector by BamHl and Xb ⁇ l (filled-in) digestion and cloned into pBluescript SK" (in which the Xbol site was removed by filling in) at the BamHl-EcoRV sites generating pSK"VIP2R-HA.
- the IR ⁇ S-/ ⁇ cZ-ADE2 cassette was isolated from pYIV2 with Notl (filled in) and Sail restriction enzymes and inserted into the Hindlll (filled in) and Sail sites of pSK ⁇ VIP2R- HA, yielding a plasmid containing VIP2R-HA-IR ⁇ S-/ ⁇ cZ-ADE2.
- the HA-IR ⁇ S-/ ⁇ cZ- ADE2 cassette was isolated by Xhol-SaH digestion and inserted into pGEMUZ at the Xh ⁇ l-SaH sites, resulting in pYIV3 (Fig.3).
- the HA-IRES- ⁇ cZ- lE>E2 cassette is flanked by Notl and Xbol restriction sites at the 5' side and by Sail and Sfil sites at the 3' side, facilitating the cloning of genomic fragments of interest for YAC manipulation.
- pYIV4 is similar to pYIV3 except that the orientation of the ADE2 gene is opposite to that in pYIV3 and two loxP elements, in same orientation, were introduced, one at the BgUl site between the lacZ and PA, and another following the ADE2 gene.
- the loxP sequence from pBG was cloned into pBluescript SK ⁇ at EcoRI and Sail sites (pSK-tecR).
- the ADE2 gene was excised from pSK " ADE2 by EcoRI and Clal (filled in) digestion and cloned into pSK ' loxP at the EcoRI and Sm ⁇ l sites (pSK"loxP- ⁇ DE2).
- the EcoRl-Sa ⁇ loxP fragment from pSK"/oxP was isolated, blunt ended with Klenow and inserted into the Bglll (filled in) site between the lacZ and poly A sequences of pSK " IR ⁇ S-/ ⁇ cZ-PA generating pSKTRES-/ ⁇ cZ-/otR-PA.
- the IRES-Z ⁇ cZ-ZoxP-PA cassette was isolated by Notl and Sail (filled in) and cloned into pSK"/ ⁇ xP- DE2 at the -VotI and BamHl (filled in) sites, yielding a construct pYIV4 (Fig.4).
- the pYF/4 vector permits deletion of the SV40 PA and AD ⁇ 2 gene in the YAC transgenic animals using Cre recombinase so that a transgene and lacZ reporter gene can be followed by its own 3'- untranslated region.
- Yeast D ⁇ A was isolated with the combined methods of Schedl et al. (26) and Bellis et al. (27). Clones were inoculated into 15 ml of medium (UraJLys-) with 2% of 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).
- 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 ⁇ -mercaptoethanol at 55°C overnight. Plugs then were washed with TE and stored at 4°C in 0.5 M EDTA.
- 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.
- pYAM4 was constructed using pYAC4, pBluescript SK" and pBG.
- pBG is a modification of pCGS990 in which the Sail site has been converted to a /Vbtl site and a PGK-Hyg-/oxP cassette has been introduced between the LYS2 and TK genes in pCGS990.
- pBG was constructed as follows: The unique Sail site in pCGS990 was converted into -V tI with the Xhol-Notl-Xhol linker
- the resulting plasmid (pHA58/ox2cm.1) was digested with BgUl, a 3.5 kb BgHl fragment harbouring a /oxP-cm-/oxP-Hyg cassette was isolated, filled in with Klenow and inserted into the EcoRI site (filled in) between the TK and LYS2 genes in pCGS990N, obtaining pCGS990N-Hygto2cm.
- the chloramphenicol resistance gene was removed from pCGS990N-Hyg/ox2cm with purified Cre recombinase. DNA was purified, transformed into E. Coli XL-1 Blue and chloramphenicol sensitive colonies were selected, leading to the production of pBG.
- a 572 bp (Smal-Clal) fragment between the cloning site and C ⁇ N4 in pYAC4 was cloned into Smal-Clal sites of pBluescript SK " vector.
- the fragment was excised with Clal and Notl and inserted into the Clal-Notl sites of pBG, resulting in pYAM3.
- a 705 bp Xhol-BamHl fragment of the telomere (TEL) from pYAC4 was blunt ended with Klenow and inserted into the filled in S ⁇ cI-S ⁇ cII sites of pBluescript SK " vector.
- pSK"7E The orientation of the TEL in the resulting plasmid was confirmed by sequencing with T7 and reverse primers.
- pSK"7EL was digested with Notl-Sall restriction enzymes and replaced the corresponding region (pBR322-7EL-7X-LoxP) in pYAM3, leading to the generation of pYAM4 (Fig. 6 ).
- pYAM4 was linearised with Notl. Yeast were inoculated into 10 ml SD medium lacking uracil and tryptophan. When yeast had grown to 2 x 10 ⁇ cells/ml, they were harvested and washed with 5 ml of LTE (0.1M LiOAc, lOmM Tris pH7.5, and 1 mM ⁇ a 2 EDTA). After resuspension in 100 ⁇ l of LTE, cells were incubated at 30°C for one hour with regular inversion. One ⁇ g of linearised pYAM4 and 5 ⁇ l of carrier DNA (salmon sperm DNA, 10 mg/ml) were added to the cells and mixed.
- LTE 0.1M LiOAc, lOmM Tris pH7.5, and 1 mM ⁇ a 2 EDTA
- 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 (UraJLys") with 2% of 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).
- 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 Na 2 EDTA, 1 % Lithium sulphate, and 0.5 M ⁇ - mercaptoethanol at 55°C overnight. Plugs then were washed with TE and stored at 4°C in 0.5 M EDTA.
- 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.
- the vector pYIV3 was used to introduce the haemagglutinin (HA) tag and lacZ reporter gene into two YAC clones,35D8 (500kb) and HSC7E526 (630kb), which contain the human serotonin transporter (SERT) and VIP2 receptor (VIPR2) genes respectively.
- SERT human serotonin transporter
- VIP2 receptor VIP2 receptor
- the X ⁇ oI site in the polylinker of pBluescript SK" was removed by digesting the vector with Xbol and filling in the recessed 3 ' termini with Klenow fragment of E. coli DNA polymerase I, generating pSKX.
- a BamHl-Xbal fragment containing the human VIPR2 cDNA with the HA tag at the C-terminus of the coding sequence was subcloned from the ⁇ cDNA3 vector into pSKX at the EcoRV site in an orientation such that the 5' end of the cDNA was adjacent to the T3 primer in the pSKX vector, generating pSK-VIPR2-HA.
- a Notl-Pstl fragment of pSK " VIPR2-HA containing VIPR2 cDNA sequences was then replaced with a 1.2 kb Notl- Pstl fragment of VIPR2 genomic DNA (Pstl cuts in the last coding exon of the human VIPR2 gene), generating pVHA.
- the IRES-/ ⁇ cZ-PA-Ade2 cassette was excised from pYIV2 as a Sail - Notl (blunt ended) fragment and inserted into the Sal -Hindlll (blunt ended) sites of pVHA, resulting in pVHAIZA.
- X ⁇ oI and Kpril restriction sites were introduced at ends of a 1.6 kb fragment of genomic DNA 3' of the stop codon of the human VIPR2 gene.
- the fragment was subcloned into the Xbol and Kp ⁇ l sites of the pSK " vector, generating p3'VIPR2.
- the Notl-Sa fragment of pVHAIZA which contains VIPR2-HA-IRES-/ ⁇ cZ-PA-Ade2 was ligated into Notl-Xh ⁇ l digested p3NIPR2, generating a final construct, pLacZVIPR2 + .
- genomic DNA sequences at least a few hundred bp in length must flank the stop codon of the target gene either side of the HA-IRES-/ ⁇ cZ-Ade2 sequences of pYIV3.
- the Xbol site in the polylinker of pBluescriptSK " was removed by digesting the vector with Xbol and filling in the recessed 3' termini with Klenow fragment of E.coli DNA polymerase, generating pSKX.
- a BamHl-Xbal fragment containing the human VIPR2 cDNA with the HA tag at the C-terminus of the coding sequence was subcloned from the 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-Xh ⁇ l sites of pBluescriptSK " using PCR primers
- the 5 kb SERT intron 13 sequence (Notl-Xh ⁇ l fragment) was used to replace the Notl-Xh ⁇ l fragment in pSK-VIPR2-HA, generating pInl3-HA.
- the intron 13 sequence and the HA tag were isolated as a S ⁇ cII - Clal (blunt ended) fragment and inserted into S ⁇ cII and (blunt ended) Notl sites of pYIV2, generating pInl3-HA-IZA.
- the sequences downstream of the stop codon in exon 14 of the SERT gene were isolated by PCR of human genomic D ⁇ A using primers 32358 (5' CTC CTC GAG AGG AAA AAG GCT TCT 3')
- the pLacZVIPR2 + and pLacZSERT + constructs were linearised with Notl and introduced into the YAC clones HSC7E526 and 35D8 respectively.
- the transformants which incorporated HA-/ ⁇ cZ-Ade2 sequences into YAC D ⁇ A by homolgous recombination were selected by growing on plates lacking uracil, tryptophan and adenine.
- the integration of the HA-IRES-/ ⁇ cZ-Ade2 sequence into the YAC D ⁇ A was confirmed by Southern hybridization with an Ade2 probe.
- YAC subclones which incorporated the HA-/ ⁇ cZ-Ade2 cassette were transformed with Notl linearised pYAM4. Recombinants were isolated on selective medium lacking uracil, adenine and lysine and replica plated on plates lacking uracil, adenine and tryptophan. Successful replacement of the YAC left arm (containing TRP1 gene) by pYAM4 would result in yeast capable of growth on medium lacking uracil, adenine and lysine but not on the counter selection medium lacking tryptophan.
- Tryptophan sensitive clones were cultured in selective medium (Ura ' /Ade /Lys ) with galactose as carbon source instead of glucose.
- selective medium Ura ' /Ade /Lys
- galactose as carbon source instead of glucose.
- the GAL1 promoter adjacent to the CE ⁇ 4 in the pYAM4 vector will be induced.
- Activation of transcription from the GALl 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.
- 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 invention where appropriate.
- 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).
- TENPA lOmM Tn's-HCI (pH 7.5), ImM EDTA (pH8.0), lOOmM NaCl, 30 ⁇ M spermine, 70 ⁇ M spermidine.
- IB lOmM Tris-HCI (pH7.5), O.lmM EDTA (pH8.0), lOOmM NaCl, 30 ⁇ M spermine, 70 ⁇ M spermidine.
- LiDS 1 % lithium-dodecyl-sulphate, lOOmM EDTA (pH8.0).
- CHEF-DR 11 pulsed-field-gel-electrophoresis (PFGE) system, BIO-RAD Laboratories, Richmond, CA, USA.
- PFGE pulsed-field-gel-electrophoresis
- the cell pellet should be about 1 to 1.5ml.
- DNA plugs prepared this way can be stored without degradation for at least one year.
- the integrity of the DNA can be checked running 20 ⁇ l of the preparation on a PFGE gel (use a comb with small slots).
- the Zeiss Automatic Injection System can be used for rapid injection of large 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 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, 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, whereas cells on confluent plates do not grow in one plane making it impossible to inject all cells into the nuclei.
- the efficiency of microinjection will depend greatly on the cell type. Best results are achieved using cells with big and easily visible nuclei.
- Liquid paraffin is preferably used to prevent contamination of the cells as well as evaporation of the medium during injections.
- STORE DATA Allows to generate a file in which the positions of the injected cells will be stored. To use this option the bottom of the dish has to be marked to give the machine left and right hand references (scratch crosses at either side). Find the marks after the plate has been place on the stage and click cursor on the appropriate box to record the references. If you generate a file you must enter an operator and a sample name. 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 most applications.
- 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 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.
- STEP DOWN Lowers the needle in the smallest possible increment.
- MARK TIP Allows to set the reference point for the computer software. To adjust click on the very tip of the injection needle.
- INJECTION TIME Determines the time the needle remains within the cell and is, therefore, one parameter for the volume delivered to the nucleus. This time has to be varied depending on the pressure, tip size etc. A time of 0.2s is a good value to start with.
- POSITION OK Click on this when you are ready to start injecting.
- MARK NEXT This will allow to direct the computer to the nuclei of cells to be injected. Click on MARK and subsequently onto the nuclei. To start the injections click on INJECT. The computer will perform the injections into the marked cells. Successfully injected cells can be identified by a temporary dramatic swelling of the nucleus. If no change of cells can be observed after a number of injections check the following possibilities:
- the injection needle is blocked: Use the high pressure button (P3) at the injection machine to release DNA. If this does not help the needle has to be replaced. •
- the computer is injecting in the wrong plane: Stop the injections by pressing the yellow button and try lowering or lifting the needle in single step increments. Be careful not to break the needle on the surface of the dish by lowering it too much. Too low pressure: Increase the pressure for PI. Be aware that too high pressure will result in bursting of the cells.
- 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 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).
- Preparation of DNA constructs for injection normally involves a filtration step in which the DNA is passed through a membrane with 0.2 ⁇ m 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 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.
- Transgenic animals can be identified by PCR or Southern blot analysis with DNA isolated from tail tips. With 250kb constructs about 10 to 20% of the offspring should have YAC DNA incorporated. Once a transgenic line has been established it is important to confirm the integrity of the integrated construct. This can be achieved by conventional PFGE mapping with several probes scattered over the YAC, which, however, requires a detailed knowledge of the restriction map of the construct. Alternatively, the RecA approach can be used to release the entire YAC from the mouse genome.
- transgenic animals of genes expressed in YACs can be greatly facilitated by the use of a reporter gene for the accurate and sensitive detection of cellular sites of transcription.
- a reporter gene for the accurate and sensitive detection of cellular sites of transcription.
- pYIVl, pYIV2, pYIV3 and pYIV4 The common feature of all of these vectors is that they contain a lacZ reporter gene downstream of a viral internal ribosome entry site (IRES), together with selective markers.
- IRS viral internal ribosome entry site
- the lacZ reporter gene will be expressed in the same pattern as the transgene so that the expression, regulation and function of the transgene can be analysed using simple histochemical staining procedures.
- the pYIVl vector can be used for YACs which have been introduced 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.
- 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 commercially available 12CA5 monoclonal antibody.
- pYIV4 contains loxP elements flanking the SV40 polyadenylation signal and the ADE2 gene.
- the poly A 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.
- Cre recombinase so that the transgene and lacZ reporter gene are flanked by the authentic 3 '-untranslated region of the transgene.
- IGF2 Insulin-like growth factor II
- 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 expressing lines produced an X-Gal (it is understood that the terms X-Gal, ⁇ -Gal and LacZ are synonymous) staining pattern (Fig.5) identical to that of the human gene from which the promoter was derived. These data demonstrated that the IRES-tocZ reporter gene is functional in the YAC insertion vectors.
- the plasmid was linearised with Notl and introduced into a variety of YAC clones from the ICI, ICRF and chromosome-7-specific YAC libraries. Recombinants were isolated on selective medium lacking uracil and lysine and replica plated on plates lacking uracil and tryptophan. Successful replacement of the left arm (containing 7RPi gene) by pYAM4 would result in yeast capable of growth on medium lacking uracil and lysine but not on the counter selection medium lacking tryptophan.
- 167 clones could not grow on medium lacking tryptophan (Table 1). That is, the homologous recombination leading to the loss of the TRPI gene occurred in 167 clones.
- the retrofitting efficiency of pYAM4 overall is 13.3% which is much higher than pCGS990 and pCGS966 (0.5-2.5%) (11, 12).
- Tryptophan sensitive clones were culmred in selective medium (UraJLys " ) with galactose as carbon source instead of glucose.
- the GALl promoter adjacent to the CEN4 in the pYAM4 vector will be induced.
- Activation of transcription from the GALl promoter should interfere with the CEN4 leading to non-segregation of the YAC DNA therefore increase the YAC DNA copy number per cell.
- 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. Introduction of an additional conditional promoter such as ADH2 adjacent to the CEN4, or of an additional selection gene, might improve the amplification further.
- YAC DNA can be introduced into mammalian cells such as embryonic stem (ES) cells, which is an alternative approach to microinjection of YAC DNA for making YAC transgenic animals (29).
- ES embryonic stem
- a genomic fragment (which is present in a YAC) is cloned into the Clal-Notl site of pYAM4, the truncation of a large YAC and amplification of the shortened YAC DNA can be achieved in a single step.
- amplification of the YAC DNA can be seen from Fig 8 and 9.
- Lane 1 is un-amplified YAC DNA as present in original 35D8 YAC clone
- lane 2 is un-amplified YAC DNA in another YAC clone (132C6) containing the SERT gene
- lane 3 is amplified YAC DNA in 35D8/D6 subclone.
- the blot was hybridized with genomic probes downstream (Fig.8) and upstream (Fig.9) of the SERT gene.
- Figure 11 is a photograph of a gel prepared by a Southern blot and hybridised with a 32 P-labelled pBR322 probe to detect YAC sequences.
- 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) and digested with gelase.
- YAC DNA was dialysed against the microinjection buffer for 2 hours before injection into fertilised oocytes.
- mice Two hundred and ninety-eight fertilised oocytes were injected with 35D8/D6 YAC DNA and 364 with HSC7E526/V12. After transfer of injected oocytes into oviducts of 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:
- N.E.I.T Number of oocytes injected and transferred PCR determination of the size of the integrated construct
- the size of the integrated YAC 35D8/D6 and YAC HSC7E526/V12 constructs in each transgenic founder animal were determined using two pairs of PCR primers (A and H: Table III) to detect the two YAC vector arms and a series of PCR primer pairs spanning the SERT (B to G: Table III) and VIPR2 (I to L: Table III) genes respectively.
- Figure 12 shows the size of the integrated YAC DNA in transgenic founder animals carrying 35D8/D6 YAC DNA.
- Figure 13 shows the size of integrated YAC DNA in transgenic founder animals carrying HSC7E526/V12 YAC DNA.
- the probable extent of 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/V12 YAC ( Figure 13) construct which are drawn above the markers.
- mice carrying the intact YAC 35D8/D6 (A102.3, A102.5, A105, Figure 12) and six carrying the intact YAC HSC7E526/V12 (A108, A108.1, A108.2, A108.3, A108.5, A110: Figure 13) were identified.
- a beneficial number of mice born in this study carried intact YAC DNA.
- mice were anaesthetised with a lethal dose of sodium pentobarbitone and briefly perfused through the heart with 0.9% sodium chloride solution to remove blood followed by a longer perfusion of the ice-cold fixative solution (4% paraformaldehyde in 0.1M sodium phospahate buffer, pH 7.4). After perfusion with approximately 150-200 ml of the fixative solution, the brains and internal organs were removed rapidly and postfixed in the same fixative for 2-4 hours at 4°C.
- the fixative solution 4% paraformaldehyde in 0.1M sodium phospahate buffer, pH 7.4
- ⁇ -galactosidase activity in transgenic mice was consistent with the published distribution of VIPR2 mRNA (Cagampang et al. , 1998; Inagaki et al., 1994; Sheward et al., 1995; Usdin et al., 1994) and of binding sites for the selective VIP2 receptor agonist Ro25-1553(Vertongen et al., 1997).
- high levels of expression of ⁇ -galactosidase were detected:
- mice Tissues from mice were dissected, frozen on dry ice and stored at -70°C. They were thawed and homogenised immediately in 100-400 ⁇ l of cold lysis buffer (as supplied in the kit, with 0.2mM PMSF and 5 ⁇ g/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 ⁇ -galactosidase. After centrifugation for 5 min at room temperature 20 ⁇ l of each sample were used in the assay. 200 ⁇ l of Galacto-Light reaction buffer was added, inbubated for 60 min at room temperature and then 300 ⁇ l of Accelerator was added and the sample counted in a TD-
- transgenes in these animals can only be assessed by in situ hybridisation, Northern blotting, PCR and/or immunohistochemistry.
- Introduction of a reporter gene into the YAC DNA would simplify procedures for the detection of transgene expression.
- YAC modification vectors pYIVl, pYIV2, pYIV3 and pYIV4 which can be inserted into YACs after the translation initiation or stop codon.
- the vectors contain a lacZ reporter gene downstream of a viral internal ribosome entry site (IRES), so that a simple histochemical staining procedure can be used to examine the tissue distribution and regulation of the transgene.
- IRS viral internal ribosome entry site
- pYIV3 permits the HA epitope tag sequence (from influenza hemagglutinin) to be fused to the carboxyl terminus of the gene product of interest, so that the protein product of the transgene can be detected using the commercially available 12CA5 monoclonal antibody.
- pYIV4 contains loxP elements flanking the SV40 polyadenylation signal and the ADE2 gene.
- poly A and ADE2 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.
- transgenic technology has played an important role in the understanding of gene function and regulation in vivo, and in creating animal models of human genetic diseases.
- yeast artificial chromosome (YAC) technology has permitted the cloning of DNA segments thousands of kilobases in size 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.
- transgenic animals of genes expressed in YACs can be greatly facilitated by the use of a reporter gene in accordance with the present invention for the accurate and sensitive detection of cellular sites of transcription.
- yeast artificial chromosome YAC
- YACs can be used to clone the complete sequences of large genes or gene complexes 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).
- bacterial artificial chromosome (BAC) (4) and PI artificial chromosome vectors (5) have a large cloning capacity (up to 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 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 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 animals.
- the efficiency with which transgenic animals are produced using YAC DNA (1-5%) is lower than that using conventional vectors (approx. 10%)
- YAC vectors for example pYAC4 contain a yeast centromere, two telomeres, and two selective markers (7RP2 and URA3). After incorporation of YAC DNA, yeast can grow in medium lacking the uracil and tryptophan. Under these conditions of selection, YAC clones are replicated along with the endogenous host chromosomes; only one copy of YAC DNA is produced per cell. Using standard protocols, YAC DNA at a concentration of 1 ⁇ g/ml can be isolated. However, there is a substantial increase in copy number if the YAC centromere is inactivated by induced transcription from a GALl or ADH2 promoter. This increase is thought to reflect the segregation bias of the YAC for the mother cells and loss of the daughter cells without the YAC from the population under selection.
- a fertilised egg would only receive 1 molecule of YAC DNA.
- Amplification of YACs in yeast therefore provides a possible method for the isolation of more concentrated YAC DNA which should lead to more successful generation of YAC transgenic animals.
- pCGS966 (11) and pCGS990 (12). Both vectors include a conditional centromere and a 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 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.
- TK thymidine kinase
- TK herpes simplex virus thymidine kinase
- YAC DNA contains a selectable marker (hygromycin B resistance) which facilitates the transfer of YAC DNA into embryonic stem cells and other cell lines.
- 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 expression pattern of the NOI.
- a NOI such as the SERT gene
- a reporter gene such as LacZ
- Transgenic mice overexpressing the human VIPR2 gene together with the ⁇ - galactosidase 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 identified: (i) agents regulating the expression of the human VIP2 receptor, which could be identified by their ability to influence ⁇ -galactosidase activity in transgenic mice and (ii) agonists and antagonists of the human VIP2 receptor, for which the transgenic mice in which the human VIPR2 gene is expressed will provide an animal model.
- YAC transgenic animals such as VIP2 receptor null (“knock out") mice, could be bred with a view to generating "humanised" animals in which the VIP2 receptor displays identical pharmacology to that seen in man.
- agents acting in the suprachiasmatic nucleus, which are capable of influencing the activity of the VIP2 receptor, may prove useful in the treatment of the disturbances in circadian function.
- Such disturbances which may lead to impaired physical and mental well-being, can occur through: (i) extremes in work patterns (shift work); (ii) travelling through many time zones (jet lag); (iii) in normal ageing; and (iv) in dementia.
- Such agents may also prove useful in the treatment of sleep disorders, seasonal affective disorder (SAD), eating disorders and pre-menstrual syndrome.
- agents, acting in the pancreas which are capable of regulating the expression of the human VIP2 receptor or agents acting as agonists and antagonists of the receptor may be useful in the treatment of diabetes.
- a YAC amplification vector such as 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.
- 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. artificial chromosomes other than YACs), which may in turn be used to prepare transgenic organisms (including animals).
- artificial chromosomes i.e. artificial chromosomes other than YACs
- transgenic organisms including animals.
- YAC represents any suitable artificial chromosome, preferably a yeast artificial chromosome.
- NCIMB National Collections of Industr ial and Marine Bacter ia Limited
- ⁇ . T.-.e muicaticn. rr._iz oeiow relate 1 0 tne mio.ourza ⁇ ism referre ⁇ to in tne esc ⁇ ptiun 1 onaace 27 line
- NCIMB N ational Collections of In d ustrial and Marine Bacteria Limited
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WO1994024301A1 (en) * | 1993-04-21 | 1994-10-27 | The University Of Edinburgh | Expression of heterologous genes according to a targeted expression profile |
WO1996040959A1 (en) * | 1995-06-07 | 1996-12-19 | Cytotherapeutics, Inc. | Cell line producing analgesic compounds for treating pain |
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EP1034257A2 (en) | 2000-09-13 |
GB9826126D0 (en) | 1999-01-20 |
GB2331752B (en) | 2001-01-24 |
GB2331752A (en) | 1999-06-02 |
CA2311282A1 (en) | 1999-06-10 |
GB2331752A9 (en) | |
AU751811B2 (en) | 2002-08-29 |
WO1999028449A3 (en) | 1999-07-29 |
JP2001525168A (en) | 2001-12-11 |
AU1342399A (en) | 1999-06-16 |
HK1019765A1 (en) | 2000-02-25 |
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