AU737409B2 - Neuronal stem cell gene - Google Patents

Description

NEURONAL STEM CELL GENE The present invention relates to a method of marking, selecting and generating neuronal stem cells from tissues. In particular, the invention relates to the use of the Soxl gene for the generation of neuroblasts.

SOX proteins constitute a family of transcription factors related to the mammalian testis determining factor SRY through homology within the HMG box DNA binding domains. In DNA binding studies, SOX proteins exhibit sequence specific binding; however, unlike most transcription factors, binding occurs in the minor groove resulting in the induction of a dramatic V. bend within the DNA helix. SOX proteins can induce transcription of reporter constructs in vitro and display properties of both classical S: 15 transcription factors and architectural components of chromatin (reviewed by Peveny and Lovell-Badge (1997) Curr. Opin. Genetics and Development, 7:338-344).

Members of the Sox gene family are expressed in a variety of embryonic and 20 adult tissues, where they appear to be responsible for the development and/or elaboration of particular cell lineages. Sry is transiently expressed in the precursor Sertoli cells of the XY genital ridge and is responsible for triggering development of the male phenotype (reviewed by Lovell-Badge and Hacker, (1995) Phil. Trans. R. Soc. Lond. B 350:205-214). Thus, the lack of Sry results 25 in XY females and XX males. Sox9 is expressed in immature chondrocytes and male gonads; mutations in the human SOX9 gene are associated with Campomelic Dysplasia, a human skeletal malformation syndrome, and XY female sex reversal. Sox4 is expressed in many tissues and a null mutation of the gene in mouse results in the absence of mature B cells and heart malformations. Xsoxl 7 genes are involved in endoderm formation in Xenopus embryos. These functional analyses suggest that Sox genes function in cell fate decisions in diverse developmental pathways.

A subfamily of Sox genes, that includes Soxl, Sox2 and Sox3, shows expression of profiles during vertebrate embryogenesis that suggest the genes could function in the control of cell fate decisions within the early developing

IS

2 nervous system. Sox2 and Sox3 begin to be expressed at preimplantation and epiblast stages respectively, and are then restricted to the neuroepithelium.

Soxl appears only at around the stage of neural induction.

The molecular mechanisms controlling neural induction and determination have begun to be elucidated. The identification by cellular and biochemical methods, of secreted molecules involved in neural induction illustrates the important role of the environment in specifying cell identity. In addition, a number of transcription factors have been isolated which play important roles in the specification and differentiation of neural cell lineages. For example, the characterisation of vertebrate homologues of Drosophila proneural and neurogenic genes, which control neural specification in the fly, has revealed analogous molecular mechanisms in vertebrate neural cell fate determination and differentiation. In Drosophila, the expression of basic helix-loop-helix 15 transcription factors of the AS-C complex confirms neural potential on groups of ectodermal cells. Miss expression of a transcription factors involved a neural cell fate determination is observed to cause abnormalities in neural development.

00*0 20 It is known that Soxl expression appears only at around the stage of neural :induction in the embryo. The role of SOX1 in embryogenesis is, however, not known.

Summary of the Invention It is shown herein that Soxl expression correlates with the formation of the neural plate. Moreover, the onset of Soxl expression in embryonal carcinoma cells is shown to be dependent on neural induction. Upregulation of Soxl expression is itself sufficient to impart a neural fate on pluripotent cells.

,1, WO 99/00516 PCT/G B98/01862 3 In a first aspect of the present invention, there is provided a method for isolating a neuroblastic cell from a population of cells comprising the steps of: detecting the expression of the Soxl gene in the cells; and sorting the cells to isolate those cells expressing the Soxl gene.

As set forth in the following description, the Soxl gene, which encodes SOX1, is responsible for the specification of neuroblast or neuronal stem cells, as well as acting as a marker for such cells. Expression of Soxl is responsible for the generation of the neuroblastic cell type, which in vivo is capable of differentiating into the many different cells and ganglia of the CNS. Moreover, Soxl is a unique marker for neuroblasts.

Cells which are identified as expressing this gene, for example by binding to anti- SOX1 antibodies, by activation of SOX1 dependent ligand-receptor systems or by detection with antisense nucleic acids specific for Soxl mRNA, are pluripotent neuroblasts. Such cells can be identified in early embryonic tissue or adult CNS material. Cells can be sorted by affinity techniques, or by cell sorting (such as fluorescence-activated cell sorting) where they are labelled with a suitable label, such as a fluorophore conjugated to or part of, for example, an antisense nucleic acid molecule or an immunoglobulin.

According to a second aspect of the invention, neuroblast cells can be actively sorted from other cell types by detecting the expression of SOX1 in vivo using a reporter system. Thus, for example, the invention provides a method for isolating a neuroblastic cell from a population of cells, comprising the steps of: transfecting the population of cells with a genetic construct comprising a coding sequence encoding a detectable marker operatively linked to the Soxl control regions; detecting the cells which express the selectable marker; and WO 99/00516 PCT/GB98/01 862 4 sorting the cells which express the selectable marker from the population of cells.

As before, the selectable marker may be any selectable entity, but is preferably a fluorescent or luminescent marker which may be detected and sorted by automated cell sorting approaches. For example, the marker may be GFP or luciferase. Other useful markers include those which are expressed in the cell membrane, thus facilitating cell sorting by affinity means.

The genetic construct according to the invention may comprise any promoter and enhancer elements as required, so long as the overall control remains sensitive to SOXI; in other words, no expression of the marker coding sequence should take place in the absence of SOX1. The regulatory sequences of the SOX1 gene are known in the art and have been described in the literature cited herein and incorporated herein by reference; 'at least, however, the construct of the invention will comprise a SOX1 binding site. Preferably, the SOX1 control elements are used in their entirety; however, other promoter and enhancer elements may be substituted where they remain under the influence of SOX1.

The selectable marker will only be expressed in neuroblastic cells because only these cells express SOX1, which is required for transcription from the Soxl control sequences. Preferably, therefore, the expression system used to express the selectable marker is not leaky and expresses a minimal amount of the marker in the absence of SOX1. Techniques for transforming cells with coding genetic constructs according to the invention, detecting the marker and sorting cells accordingly are known in the art.

The present invention, in a third aspect, provides the use of the Soxl coding sequence to transform precursor cells and thereby differentiate neuroblast cells therefrom. Accordingly, there is provided a method for differentiating one or more WO 99/00516 PCT/GB98/01862 neuroblastic cells from one or mores pluripotent precursor cell, comprising the steps of: transforming the pluripotent precursor cell(s) with a genetic construct comprising a Soxl coding sequence operatively linked to suitable control sequences; and culturing the cell(s) so as to allow expression of the Soxl coding sequence, thereby inducing the cell to differentiate into a neuroblast.

Suitable control sequences for use in the third aspect of the invention are known in the art and may include inducible or constitutive control sequences. Inducible control sequences have the advantage that Soxl expression may be switched off when desired, for example once the cell is to be differentiated into another neural cell. Moreover, once the expression of exogenous Soxl has been switched off, successfully differentiated neuroblasts may be identified by virtue of the continued expression of the endogenous Soxl gene.

Precursor cells may be, for example, ES cells, such as human ES cells and cells with similar pluripotent properties derived from germ cells (EG cells). More specific neuronal pluripotent precursors or direct neuroblast precursors may also be employed.

Neuroblasts obtained according to the invention may be employed in a number of ways. Of course, the expression of Soxl has important implications for the study of neural differentiation: the generation and selection of neuroblasts will provide material for basic research.

Moreover, the invention has medical and diagnostic applications. The detection of Soxl expressing cells is important in clinical neurology and in diagnosing and treating cancers of the nervous system. Accordingly, the invention provides a method for detecting the presence of a neuroblast as described above for diagnostic purposes.

WO 99/00516 PCT/GB98/01862 6 Neural stem cells are also useful for the treatment of neurological disorders, especially for repair of accidentally induced trauma in the CNS or for the correction of congenital or pathological diseases of the CNS.

Moreover, in applications involving somatic gene therapy designed to correct a genetic defect in nervous tissue, the removal, treatment and replacement of pluripotent neuroblasts which are actively dividing has clear advantages, providing a constant source of modified neural cells to permanently treat the targeted defect.

Soxl control sequences may be used specifically to direct transgene expression in neuroblast cells where this is desired. Moreover, gene expression can be directed to neural cell types differentiated from neuroblasts by the use of other control sequences, such as NF-1 control sequences which direct expression of NF-1 in mature neurons in vivo.

A significant advantage of the methods described herein is that a patient in need of treatment for a neurological disorder can act as a self-donor. In other words, cells may be isolated from the patient and either sorted to extract neuroblasts, or treated in order to differentiate neuroblasts as described, from specific or general precursors.

Detailed Description of the Invention The present invention relates to a method for isolating, or producing cells which are committed to the neural fate. Accordingly, the term neuroblast, as used herein, refers to any cell or cell line which has commenced differentiation along the neural pathways.

The isolation of neuroblastic cells from populations of cells is desirable, in order to obtain cells which are committed to neural pathways, but are not terminally differentiated. Such cells are useful in the study of neuronal differentiation, and in WO 99/00516 PCT/GB98/01862 7 the treatment of diseases such as neurodegenerative diseases, and neural damage, for example occasioned by trauma. Thus, typical populations of cells from which neuroblastic cells may be differentiated include cell populations derived from the CNS of mammals, such as humans, including CNS from adult and foetal sources.

Moreover, cell populations derived from tissue cultures may be employed for the isolation of neuroblastic cells.

It has been determined that SOX1 expression is closely associated with the acquisition of neural fate by the ectoderm, both in vitro and in vivo. In vitro SOX1 expression is initiated within 24 hours after the addition of retinoic acid to pluripotent EC cell aggregates coincident with the induction of neuroepithelial markers such as NESTIN, Mash1 and Wntl. In mouse and rat embryos expression is restricted to cells of the antero/distal ectoderm. Previous fate mapping studies indicated that this region of the epiblast constitutes the promordium of the nervous system.

Expression of SOX1 is detected throughout the cells of the neural plate and early neural tube along its entire anteroposterior axis. The early and uniform expression of SOX1 throughout the presumptive CNS indicates that SOX1 is activated by neural inducing signals and lends support to the proposal of a two step response of the ectoderm to organiser signals in generating a nervous system: neutralisation followed by regionalisation.

Expression of this Sox gene subfamily has been evolutionarily conserved. The Drosophila (Nambu and Nambu 1996; Russel et al., 1996) zebrafish (Vriz et al., 1996) and avian (Unwanogho et al., 1995; Streit et al., 1997; Rex et al., 1997) putative orthologues of Soxl, Sox2 and Sox3 all show expression throughout the neural primordium. Thus, this subfamily of Sox genes represents a novel group of transcription factors which can serve as general early neuroepithelial markers.

WO 99/00516 PCT/GB98/01862 8 In order to isolate neuroblastic cells, the present invention provides for the detection of Soxl therein. As used herein, Soxl may be derived from any source, including mammalian sources, avian sources and other vertebrate sources. Soxl may also be derived from invertebrate sources.

Soxl has been cloned from human, chicken and mouse. The sequence of chicken, mouse and human Soxl is set forth in SEQ ID.s numbers 1 to 3 herein.

The preferred sequence encoding Soxl is that encoding human Soxl and having substantially the same nucleotide sequence as the sequence in SEQ ID No. 3, with the nucleic acid having the same sequence as the sequence in SEQ ID No. 3 being most preferred. As used herein, nucleotide sequences which are substantially the same share at least about 90% identity. However, in the case of splice variants having e.g. an additional exon sequence homology may be lower.

The nucleic acids of the invention, whether used as probes or otherwise, are preferably substantially homologous to the sequence of human Soxl as shown in SEQ ID No. 3. As used herein, "homology" means that the two entities share sufficient characteristics for the skilled person to determine that they are similar in origin and function. Preferably, homology is used to refer to sequence identity.

Thus, Soxl sequences according to the invention preferably retain substantial sequence identity human Soxl.

"Substantial homology", where homology indicates sequence identity, means more than 40% sequence identity, preferably more than 45% sequence identity and most preferably a sequence identity of 50% or more, as judged by direct sequence alignment and comparison.

Sequence homology (or identity) may be determined using any suitable homology algorithm, using for example default parameters. Advantageously, the BLAST algorithm is employed, with parameters set to default values. The BLAST WO 99/00516 PCT/GB98/01862 9 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, and are advantageously set to the defined default parameters.

Advantageously, "substantial homology" when assessed by BLAST equates to sequences which match with an EXPECT value of at least 7, preferablyat least 9 and most preferably 10 or more. The default threshold for EXPECT in BLAST searching is ususally BLAST (Basic Local Alignment Search Tool) is the heuristic search algorithm employed by the programs blastp, blastn, blastx, tblastn, and tblastx; these programs ascribe significance to their findings using the statistical methods of Karlin and Altschul (see http://www.ncbi.nih.gov/BLAST/blast_help.html) with a few enhancements. The BLAST programs were tailored for sequence similarity searching, for example to identify homologues to a query sequence. The programs are not generally useful for motif-style searching. For a discussion of basic issues in similarity searching of sequence databases, see Altschul et al. (1994) Nature Genetics 6:119-129.

The five BLAST programs available at http://www.ncbi.nlm.nih.gov perform the following tasks: blastp compares an amino acid query sequence against a protein sequence database; blastn compares a nucleotide query sequence against a nucleotide sequence database; blastx compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database; WO 99/00516 PCT/GB98/01862 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 1o 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). See also EXPECT and CUTOFF.

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).

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).

WO 99/00516 PCT/GB98/01862 11 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.

MATRIX Specify an alternate scoring matrix for BLASTP, BLASTX, TBLASTN and TBLASTX. The default matrix is BLOSUM62 (Henikoff Henikoff, 1992).

The valid alternative choices include: PAM40, PAM120, PAM250 and IDENTITY.

No alternate scoring matrices are available for BLASTN; specifying the MATRIX directive in BLASTN requests returns an error response.

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

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

Low complexity sequence found by a filter program is substituted using the letter in nucleotide sequence "NNNNNNNNNNNNN") and the letter in WO 99/00516 PCT/GB98/01862 12 protein sequences "XXXXXXXXX"). Users may turn off filtering by using the "Filter" option on the "Advanced options for the BLAST server" page.

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

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

NCBI-gi Causes NCBI gi identifiers to be shown in the output, in addition to the accession and/or locus name.

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

Preferably, the invention makes use of fragments of the Soxl-encoding sequence.

Fragments of the nucleic acid sequence of a few nucleotides in length, preferably to 150 nucleotides in length, are especially useful as probes.

Exemplary nucleic acids can alternatively be characterised as those nucleotide sequences which encode a Soxl protein and hybridise to the DNA sequences set forth SEQ ID No. 3, or a selected fragment of said DNA sequence. Preferred are such sequences encoding Soxl which hybridise under high-stringency conditions to the sequence of SEQ ID No. 3.

Stringency of hybridisation refers to conditions under which polynucleic acids hybrids are stable. Such conditions are evident to those of ordinary skill in the field.

WO 99/00516 PCT/GB98/01862 13 As known to those of skill in the art, the stability of hybrids is reflected in the melting temperature (Tm) of the hybrid which decreases approximately 1 to with every 1% decrease in sequence homology. In general, the stability of a hybrid is a function of sodium ion concentration and temperature. Typically, the hybridisation reaction is performed under conditions of higher stringency, followed by washes of varying stringency.

As used herein, high stringency refers to conditions that permit hybridisation of only those nucleic acid sequences that form stable hybrids in 1 M Na+ at 65-68 OC.

High stringency conditions can be provided, for example, by hybridisation in an aqueous solution containing 6x SSC, 5x Denhardt's, 1 SDS (sodium dodecyl sulphate), 0.1 Na+ pyrophosphate and 0.1 mg/ml denatured salmon sperm DNA as non specific competitor. Following hybridisation, high stringency washing may be done in several steps, with a final wash (about 30 min) at the hybridisation temperature in 0.2 0. x SSC, 0.1 SDS.

Moderate stringency refers to conditions equivalent to hybridisation in the above described solution but at about 60-62 0 C. In that case the final wash is performed at the hybridisation temperature in Ix SSC, 0.1 SDS.

Low stringency refers to conditions equivalent to hybridisation in the above described solution at about 50-52 0 C. In that case, the final wash is performed at the hybridisation temperature in 2x SSC, 0.1 SDS.

It is understood that these conditions may be adapted and duplicated using a variety of buffers, e.g. formamide-based buffers, and temperatures. Denhardt's solution and SSC are well known to those of skill in the art as are other suitable hybridisation buffers (see, e.g. Sambrook, et al., eds. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York or Ausubel, et al., eds. (1990) Current Protocols in Molecular Biology, John Wiley Sons, WO 99/00516 PCT/GB98/01862 14 Inc.). Optimal hybridisation conditions have to be determined empirically, as the length and the GC content of the hybridising pair also play a role.

Advantageously, the invention moreover provides nucleic acid sequence which are capable of hybridising, under stringent conditions, to a fragment of SEQ. ID. No. 3.

Preferably, the fragment is between 15 and 50 bases in length. Advantageously, it is about 25 bases in length.

As will be appreciated by those skilled in the art, the redundancy of the genetic code allows the design of a large number of sequences encoding human Soxl. Any of these sequences may be useful for expressing SOX1 as described below. An advantage of the use of a sequence encoding human SOX1 which is not the human Soxl sequence is that the mRNA produced has a different sequence to that of the endogenous Soxl mRNA, and may thus be distinguished therefrom. Antisense oligonucleotides may be designed which are capable of selectively inhibiting the expression of either endogenous or exogenous Soxl genes. Degenerate sequences encoding human SOX1 are set forth in SEQ. ID. No. Given the guidance provided herein, nucleic acids encoding Soxl are obtainable according to methods well known in the art. For example, a nucleic acid encoding Soxl is obtainable by chemical synthesis, using polymerase chain reaction (PCR) or by screening a genomic library or a suitable cDNA library prepared from a source believed to possess Soxl and to express it at a detectable level.

Chemical methods for synthesis of a nucleic acid of interest are known in the art .and include triester, phosphite, phosphoramidite and H-phosphonate methods, PCR and other autoprimer methods as well as oligonucleotide synthesis on solid supports.

These methods may be used if the entire nucleic acid sequence of the nucleic acid is known, or the sequence of the nucleic acid complementary to the coding strand is available. Alternatively, if the target amino acid sequence is known, one may infer WO 99/00516 PCT/GB98/01862 potential nucleic acid sequences using known and preferred coding residues for each amino acid residue.

An alternative means to isolate a gene encoding Soxl is to use PCR technology as described e.g. in section 14 of Sambrook et al., 1989. This method requires the use of oligonucleotide probes that will hybridise to Soxl nucleic acid. Strategies for selection of oligonucleotides are described below.

Libraries are screened with probes or analytical tools designed to identify the gene of interest or the protein encoded by it. For cDNA expression libraries suitable means include monoclonal or polyclonal antibodies that recognise and specifically bind to Soxl; oligonucleotides of about 20 to 80 bases in length that encode known or suspected Soxl cDNA from the same or different species; and/or complementary or homologous cDNAs or fragments thereof that encode the same or a hybridising gene. Appropriate probes for screening genomic DNA libraries include, but are not limited to oligonucleotides, cDNAs or fragments thereof that encode the same or hybridising DNA; and/or homologous genomic DNAs or fragments thereof.

A nucleic acid encoding Soxl may be isolated by screening suitable cDNA or genomic libraries under suitable hybridisation conditions with a probe, i.e. a nucleic acid disclosed herein including oligonucleotides derivable from the sequences set forth in SEQ ID NO. 3. Suitable libraries are commercially available or can be prepared e.g. from cell lines, tissue samples, and the like.

As used herein, a probe is e.g. a single-stranded DNA or RNA that has a sequence of nucleotides that includes between 10 and 50, preferably between 15 and 30 and most preferably at least about 20 contiguous bases that are the same as (or the complement of) an equivalent or greater number of contiguous bases set forth in SEQ ID No. 3. The nucleic acid sequences selected as probes should be of sufficient length and sufficiently unambiguous so that false positive results are minimised. The nucleotide sequences are usually based on conserved or highly WO 99/00516 PCT/GB98/01862 16 homologous nucleotide sequences or regions of Soxl. The nucleic acids used as probes may be degenerate at one or more positions. The use of degenerate oligonucleotides may be of particular importance where a library is screened from a species in which preferential codon usage in that species is not known.

Preferred regions from which to construct probes include 5' and/or 3' coding sequences, sequences predicted to encode ligand binding sites, and the like. For example, either the full-length cDNA clone disclosed herein or fragments thereof can be used as probes. Preferably, nucleic acid probes of the invention are labelled with suitable label means for ready detection upon hybridisation. For example, a suitable label means is a radiolabel. The preferred method of labelling a DNA fragment is by incorporating a32P dATP with the Klenow fragment of DNA polymerase in a random priming reaction, as is well known in the art.

Oligonucleotides are usually end-labelled with y 3 2 P-labelled ATP and polynucleotide kinase. However, other methods non-radioactive) may also be used to label the fragment or oligonucleotide, including e.g. enzyme labelling, fluorescent labelling with suitable fluorophores and biotinylation.

After screening the library, e.g. with a portion of DNA including substantially the entire Soxl-encoding sequence or a suitable oligonucleotide based on a portion of said DNA, positive clones are identified by detecting a hybridisation signal; the identified clones are characterised by restriction enzyme mapping and/or DNA sequence analysis, and then examined, e.g. by comparison with the sequences set forth herein, to ascertain whether they include DNA encoding a complete Soxl if they include translation initiation and termination codons). If the selected clones are incomplete, they may be used to rescreen the same or a different library to obtain overlapping clones. If the library is genomic, then the overlapping clones may include exons and introns. If the library is a cDNA library, then the overlapping clones will include an open reading frame. In both instances, complete clones may be identified by comparison with the DNAs and deduced amino acid sequences provided herein.

WO 99/00516 PCT/GB98/01862 17 It is envisaged that Sox 1-encoding sequences can be readily modified by nucleotide substitution, nucleotide deletion, nucleotide insertion or inversion of a nucleotide stretch, and any combination thereof. Such mutants can be used e.g. to produce a SOXI mutant that has an amino acid sequence differing from the SOX1 sequences as found in nature. Mutagenesis may be predetermined (site-specific) or random. A mutation which is not a silent mutation must not place sequences out of reading frames and preferably will not create complementary regions that could hybridise to produce secondary mRNA structure such as loops or hairpins.

Sorting of cells, based upon detection of expression of the Soxl gene, may be performed by any technique known in the art, as exemplified above. For example, the cells may be sorted by flow cytometry or FACS. For a general reference, see Flow Cytometry and Cell Sorting: A Laboratory Manual (1992) A. Radbruch Springer Laboratory, New York.

Flow cytometry is a powerful method for studying and purifying cells. It has found wide application, particularly in immunology and cell biology: however, the capabilities of the FACS can be applied in many other fields of biology. The acronym F.A.C.S. stands for Fluorescence Activated Cell Sorting, and is used interchangeably with "flow cytometry". The principle of FACS is that individual cells, held in a thin stream of fluid, are passed through one or more laser beams, causing light to be scattered and fluorescent dyes to emit light at various frequencies. Photomultiplier tubes (PMT) convert light to electrical signals, which are interpreted by software to generate data about the cells. Sub-populations of cells with defined characteristics can be identified and automatically sorted from the suspension at very high purity 100%).

FACS machines collect fluorescence signals in one to several channels corresponding to different laser excitation and fluorescence emission wavelengths.

Fluorescent labelling allows the investigation of many aspects of cell structure and WO 99/00516 PCT/GB98/01862 18 function. The most widely used application is immunofluorescence: the staining of cells with antibodies conjugated to fluorescent dyes such as fluorescein and phycoerythrin. This method is often used to label molecules on the cell surface, but antibodies can also be directed at targets within the cell. In direct immunofluorescence, an antibody to a particular molecule, the SOX1 polypeptide, is directly conjugated to a fluorescent dye. Cells can then be stained in one step. In indirect immunofluorescence, the primary antibody is not labelled, but a second fluorescently conjugated antibody is added which is specific for the first antibody: for example, if the anti-SOX1 antibody is a mouse IgG, then the second antibody could be a rat or rabbit antibody raised against mouse IgG.

FACS can be used to measure gene expression in cells transfected with recombinant DNA encoding SOX1. This can be achieved directly, by labelling of the protein product, or indirectly by using a reporter gene in the construct. Examples of reporter genes are p-galactosidase and Green Fluorescent Protein (GFP). pgalactosidase activity can be detected by FACS using fluorogenic substrates such as fluorescein digalactoside (FDG). FDG is introduced into cells by hypotonic shock, and is cleaved by the enzyme to generate a fluorescent product, which is trapped within the cell. One enzyme can therefore generate a large amount of fluorescent product. Cells expressing GFP constructs will fluoresce without the addition of a substrate. Mutants of GFP are available which have different excitation frequencies, but which emit fluorescence in the same channel. In a two-laser FACS machine, it is possible to distinguish cells which are excited by the different lasers and therefore assay two transfections at the same time.

Alternative means of cell sorting may also be employed. For example, the invention comprises the use of nucleic acid probes complementary to Soxl mRNA.

Such probes can be used to identify cells expressing Soxl individually, such that they may subsequently be sorted either manually, or using FACS sorting. Nucleic acid probes complementary to Soxl mRNA may be prepared according to the WO 99/00516 PCT/GB98/01862 19 teaching set forth above, using the general procedures as described by Sambrook et al (1989).

In a preferred embodiment, the invention comprises the use of an antisense nucleic acid molecule, complementary to Soxl mRNA, conjugated to a fluorophore which may be used in FACS cell sorting.

Suitable imaging agents for use with FACS may be delivered to the cells by any suitable technique, including simple exposure thereto in cell culture, delivery of transiently expressing nucleic acids by viral or non- viral vector means, liposomemediated transfer of nucleic acids or imaging agents, and the like.

The invention, in certain embodiments, includes antibodies specifically recognising and binding to SOX1. For example, such antibodies may be generated against the SOX1 having the amino acid sequences set forth in SEQ ID No. 4. Alternatively, SOX1 or SOX1 fragments (which may also be synthesised by in vitro methods) are fused (by recombinant expression or an in vitro peptidyl bond) to an immunogenic polypeptide and this fusion polypeptide, in turn, is used to raise antibodies against a SOX1 epitope.

Anti-SOX1 antibodies may be recovered from the serum of immunised animals.

Monoclonal antibodies may be prepared from cells from immunised animals in the conventional manner.

The antibodies of the invention are useful for identifying SOX1 in neural cells expressing Soxl, in accordance with the present invention.

Antibodies according to the invention may be whole antibodies of natural classes, such as IgE and IgM antibodies, but are preferably IgG antibodies. Moreover, the invention includes antibody fragments, such as Fab, F(ab')2, Fv and ScFv. Small fragments, such Fv and ScFv, possess advantageous properties for diagnostic and WO 99/00516 PCT/GB98/01862 therapeutic applications on account of their small size and consequent superior tissue distribution.

The antibodies may comprise a label. Especially preferred are labels which allow the imaging of the antibody in neural cells in vivo. Such labels may be radioactive labels or radioopaque labels, such as metal particles, which are readily visualisable within tissues. Moreover, they may be fluorescent labels or other labels which are visualisable in tissues and which may be used for cell sorting.

1o Recombinant DNA technology may be used to improve the antibodies of the invention. Thus, chimeric antibodies may be constructed in order to decrease the immunogenicity thereof in diagnostic or therapeutic applications. Moreover, immunogenicity may be minimised by humanising the antibodies by CDR grafting [see European Patent Application 0 239 400 (Winter)] and, optionally, framework modification.

Antibodies according to the invention may be obtained from animal serum, or, in the case of monoclonal antibodies or fragments thereof, produced in cell culture.

Recombinant DNA technology may be used to produce the antibodies according to established procedure, in bacterial or preferably mammalian cell culture. The selected cell culture system preferably secretes the antibody product.

Therefore, the present invention includes a process for the production of an antibody according to the invention comprising culturing a host, e.g. E. coli or a mammalian cell, which has been transformed with a hybrid vector comprising an expression cassette comprising a promoter operably linked to a first DNA sequence encoding a signal peptide linked in the proper reading frame to a second DNA sequence encoding said protein, and isolating said protein.

Multiplication of hybridoma cells or mammalian host cells in vitro is carried out in suitable culture media, which are the customary standard culture media, for example WO 99/00516 PCT/GB98/01862 21 Dulbecco's Modified Eagle Medium (DMEM) or RPMI 1640 medium, optionally replenished by a mammalian serum, e.g. foetal calf serum, or trace elements and growth sustaining supplements, e.g. feeder cells such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages, 2-aminoethanol, insulin, transferrin, low density lipoprotein, oleic acid, or the like. Multiplication of host cells which are bacterial cells or yeast cells is likewise carried out in suitable culture media known in the art, for example for bacteria in medium LB, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2 x YT, or M9 Minimal Medium, and for yeast in medium YPD, YEPD, Minimal Medium, or Complete Minimal Dropout Medium.

In vitro production provides relatively pure antibody preparations and allows scaleup to give large amounts of the desired antibodies. Techniques for bacterial cell, yeast or mammalian cell cultivation are known in the art and include homogeneous suspension culture, e.g. in an airlift reactor or in a continuous stirrer reactor, or immobilised or entrapped cell culture, e.g. in hollow fibres, microcapsules, on agarose microbeads or ceramic cartridges.

Large quantities of the desired antibodies can also be obtained by multiplying mammalian cells in vivo. For this purpose, hybridoma cells producing the desired antibodies are injected into histocompatible mammals to cause growth of antibodyproducing tumours. Optionally, the animals are primed with a hydrocarbon, especially mineral oils such as pristane (tetramethyl-pentadecane), prior to the injection. After one to three weeks, the antibodies are isolated from the body fluids of those mammals. For example, hybridoma cells obtained by fusion of suitable myeloma cells with antibody-producing spleen cells from Balb/c mice, or transfected cells derived from hybridoma cell line Sp2/0 that produce the desired antibodies are injected intraperitoneally into Balb/c mice optionally pre-treated with pristane, and, after one to two weeks, ascitic fluid is taken from the animals.

WO 99/00516 PCT/GB98/01862 22 The cell culture supernatants are screened for the desired antibodies, preferentially by immunofluorescent staining of cells expressing SOX1, by immunoblotting, by an enzyme immunoassay, e.g. a sandwich assay or a dot-assay, or a radio immunoassay.

For isolation of the antibodies, the immunoglobulins in the culture supernatants or in the ascitic fluid may be concentrated, e.g. by precipitation with ammonium sulphate, dialysis against hygroscopic material such as polyethylene glycol, filtration through selective membranes, or the like. If necessary and/or desired, the antibodies are purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose and/or (immuno-)affinity chromatography, e.g. affinity chromatography with SOX1 protein or with Protein-A.

The invention further concerns hybridoma cells secreting the monoclonal antibodies of the invention. The preferred hybridoma cells of the invention are genetically stable, secrete monoclonal antibodies of the invention of the desired specificity and can be activated from deep-frozen cultures by thawing and recloning.

The invention also concerns a process for the preparation of a hybridoma cell line secreting monoclonal antibodies directed SOX1, characterised in that a suitable mammal, for example a Balb/c mouse, is immunised with purified SOX1 protein, an antigenic carrier containing purified SOX1 or with cells bearing SOX1, antibodyproducing cells of the immunised mammal are fused with cells of a suitable myeloma cell line, the hybrid cells obtained in the fusion are cloned, and cell clones secreting the desired antibodies are selected. For example spleen cells of Balb/c mice immunised with cells bearing SOX1 are fused with cells of the myeloma cell line PAI or the myeloma cell line Sp2/0-Agl4, the obtained hybrid cells are screened for secretion of the desired antibodies, and positive hybridoma cells are cloned.

WO 99/00516 PCT/GB98/01862 23 Preferred is a process for the preparation of a hybridoma cell line, characterised in that Balb/c mice are immunised by injecting subcutaneously and/or intraperitoneally between 10 and 107 and 108 cells of human tumour origin which express SOXI containing a suitable adjuvant several times, e.g. four to six times, over several months, e.g. between two and four months, and spleen cells from the immunised mice are taken two to four days after the last injection and fused with cells of the myeloma cell line PAI in the presence of a fusion promoter, preferably polyethylene glycol. Preferably the myeloma cells are fused with a three- to twentyfold excess of spleen cells from the immunised mice in a solution containing about 30 to about 50 polyethylene glycol of a molecular weight around 4000. After the fusion the cells are expanded in suitable culture media as described hereinbefore, supplemented with a selection medium, for example HAT medium, at regular intervals in order to prevent normal myeloma cells from overgrowing the desired hybridoma cells.

The invention also concerns recombinant DNAs comprising an insert coding for a heavy chain variable domain and/or for a light chain variable domain of antibodies directed to the extracellular domain of SOX1 as described hereinbefore. By definition such DNAs comprise coding single stranded DNAs, double stranded DNAs consisting of said coding DNAs and of complementary DNAs thereto, or these complementary (single stranded) DNAs themselves.

Furthermore, DNA encoding a heavy chain variable domain and/or for a light chain variable domain of antibodies directed SOX1 can be enzymatically or chemically synthesised DNA having the authentic DNA sequence coding for a heavy chain variable domain and/or for the light chain variable domain, or a mutant thereof. A mutant of the authentic DNA is a DNA encoding a heavy chain variable domain and/or a light chain variable domain of the above-mentioned antibodies in which one or more amino acids are deleted or exchanged with one or more other amino acids. Preferably said modification(s) are outside the CDRs of the heavy chain variable domain and/or of the light chain variable domain of the antibody. Such a WO 99/00516 PCT/GB98/01862 24 mutant DNA is also intended to be a silent mutant wherein one or more nucleotides are replaced by other nucleotides with the new codons coding for the same amino acid(s). Such a mutant sequence is also a degenerated sequence. Degenerated sequences are degenerated within the meaning of the genetic code in that an unlimited number of nucleotides are replaced by other nucleotides without resulting in a change of the amino acid sequence originally encoded. Such degenerated sequences may be useful due to their different restriction sites and/or frequency of particular codons which are preferred by the specific host, particularly E. coli, to obtain an optimal expression of the heavy chain murine variable domain and/or a l0 light chain murine variable domain.

The term mutant is intended to include a DNA mutant obtained by in vitro mutagenesis of the authentic DNA according to methods known in the art.

For the assembly of complete tetrameric immunoglobulin molecules and the expression of chimeric antibodies, the recombinant DNA inserts coding for heavy and light chain variable domains are fused with the corresponding DNAs coding for heavy and light chain constant domains, then transferred into appropriate host cells, for example after incorporation into hybrid vectors.

The invention therefore also concerns recombinant DNAs comprising an insert coding for a heavy chain murine variable domain of an antibody directed SOX1 fused to a human constant domain g, for example yl, y2, y3 or y4, preferably yl or y4. Likewise the invention concerns recombinant DNAs comprising an insert coding for a light chain murine variable domain of an antibody directed to SOX1 fused to a human constant domain K or X, preferably K.

In another embodiment the invention pertains to recombinant nucleic acids wherein the heavy chain variable domain and the light chain variable domain are linked by way of a DNA insert coding for a spacer group, optionally comprising a signal sequence facilitating the processing of the antibody in the host cell and/or a DNA WO 99/00516 PCT/GB98/01862 coding for a peptide facilitating the purification of the antibody and/or a DNA coding for a cleavage site and/or a DNA coding for a peptide spacer and/or a DNA coding for an effector molecule, such as a label.

According to a further aspect, and as referred to above, neuroblastic cells may be actively sorted from other cell types by detecting Soxl expression in vivo using a reporter system. For example, such a reporter system may comprise a readily identifiable marker under the control of a Soxl activated expression system.

Fluorescent markers, which can be detected and sorted by FACS, are preferred.

Especially preferred are GFP and luciferase.

Alternatively, an in vivo construct expressing a reporter may be placed under the control of the Soxl control sequences themselves. These sequences are activated at the same time as Soxl expression is activated, and therefore mark the transition into the neural pathway with the same accuracy as Soxl. Advantageously, the Soxl control sequences used are human Soxl control sequences. Preferably, they comprise nucleotides 1 to 60 of SEQ. ID. No. 3.

In general, reporter constructs useful for detecting neural cells by expression of a reporter gene may be constructed the general teaching of Sambrook et al (1989).

Typically, constructs according to the invention comprise a promoter by Soxl, and a coding sequence encoding the desired reporter constructs, for example of GFP or luciferase. Vectors encoding GFP and luciferase are known in the art and available commercially.

SOX proteins bind to a sequence motif (A/T A/T CAA A/T G) (SEQ. ID. No. 6) with high affinity. Accordingly, constructs according to the invention advantageously comprise the above-recited motif, or a functional equivalent thereof, operably linked to a gene encoding a selectable marker.

WO 99/00516 PCT/GB98/01862 26 When transfected into cells which are potentially express Soxl, constructs according to the invention will be activated specifically by Soxl expression. Therefore, the selectable marker will be expressed once the cell enters the neural differentiation pathway and Soxl expression is induced. This allows cells entering the neural differentiation pathway to be sorted by FACS.

In a still further aspect, the present invention relates to the transfection of pluripotent precursor cells, capable of differentiating into neural cells, with a vector expressing Soxl. By such means, pluripotent precursor cells may be induced to differentiate along the neural pathway, becoming precursor neurons capable of differentiating into a variety of neural tissues.

Herein, terms such as "transfection", "transformation" and the like are not intended to be significant, except to indicate that nucleic acid is transferred to a cell or organism in functional form. Such terms include various means of transferring nucleic acids to cells, including transfection with CaPO 4 electroporation, viral transduction, lipofection, delivery using liposomes and other delivery vehicles, biolistics and the like.

Suitable pluripotent precursor cells may be derived from a number of sources. For example, ES cells, such as human ES cells and cells derived from a Germ cells (EG cells) may be derived from embryonal tissue. Alternatively, pluripotent cells may be prepared by a retrodifferentiation, by the administration of growth factors or otherwise (see WO 96/23870), or by cloning, such as by nuclear transfer from an adult cell to a pluripotent cell such as an ovum.

Human stem cells of neural lineage may be isolated from human tissues directly.

Alternatively, stem sells from non- human animals, such as rodents, may be used.

Neural stem cells may also be propagated in vitro, for example as described in Snyder et al. (1996) Clinical Neuroscience 3: 310-316, and Martinez-Serrano et al., WO 99/00516 PCT/GB98/01862 27 (1996) Clinical Neuroscience 3:301-309. Moreover, pluripotent cell lines such as the N-Tera II cell line which are capable of differentiating into neural cells upon stimulation with agents such as retinoic acid are also responsive to Soxl stimulation.

The cDNA or genomic DNA encoding native or mutant SOX1, or a label under to control of Soxl sequences or a sequence transactivatable by SOX1, can be incorporated into vectors according too techniques known in the art. As used herein, vector (or plasmid) refers to discrete elements that are used to introduce heterologous DNA into cells for expression. Selection and use of such vehicles are to well within the skill of the artisan. The vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, a transcription termination sequence and a signal sequence.

Most expression vectors are shuttle vectors, i.e. they are capable of replication in at least one class of organisms but can be transfected into another class of organisms for expression. For example, a vector is cloned in E. coli and then the same vector is transfected into mammalian cells even though it is not capable of replicating independently of the host cell chromosome.

Advantageously, an expression and cloning vector may contain a selection gene, also referred to as selectable marker, other than that intended for marking Soxlexpressing cells. This gene may encode a protein necessary for the survival of growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available from complex media.

WO 99/00516 PCT/GB98/01862 28 Since the replication of vectors is conveniently done in E. coli, an E. coli genetic marker and an E. coli origin of replication are advantageously included. These can be obtained from E. coli plasmids, such as pBR322, Bluescript vector or a pUC plasmid, e.g. pUC18 or pUC19, which contain both E. coli replication origin and E. coli genetic marker conferring resistance to antibiotics, such as ampicillin.

Expression vectors usually contain a promoter that is recognised by the host organism and is operably linked to SOX1, or label-encoding, nucleic acid. Such a promoter may be inducible by factors which induce Soxl, or by Soxl itself. The promoters are operably linked to DNA encoding SOX1 by removing the promoter from the source DNA and inserting the isolated promoter sequence into the vector.

Both the native SOX1 promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of SOX1 DNA. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

Control sequences, comprising a promoter and optionally enhancer(s), may be derived from the human or other Soxl genes. Alternatively, any suitable promoter may be used, when placed under the control of a SOX1-inducible element. In such a construct, the promoter selected should have a low residual level of activity, such as to minimise expression of the label in the absence of Soxl expression.

The vectors may also contain sequences necessary for the termination of transcription and for stabilising the mRNA. Such sequences are commonly available from the 5' and 3' untranslated regions of eukaryotic or viral DNAs or cDNAs.

These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding SOX1 or the label.

WO 99/00516 PCT/GB98/01862 29 An expression vector includes any vector capable of expressing SOX1 or labelencoding nucleic acids that are operatively linked with regulatory sequences, such as promoter regions, that are capable of expression of such DNAs. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector, that upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those with ordinary skill in the art and include those that are replicable in eukaryotic and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome. For example, DNAs encoding SOXI may be inserted into a vector suitable for expression of cDNAs in mammalian cells, e.g. a CMV enhancer-based vector such as pEVRF (Matthias, et al., (1989) NAR 17, 6418).

Particularly useful for practising the present invention are expression vectors that provide for the transient expression of DNA encoding SOX1 or a label in mammalian cells. Transient expression usually involves the use of an expression vector that is able to replicate efficiently in a host cell, such that the host cell accumulates many copies of the expression vector, and, in turn, synthesises high levels of SOXlor a label. For the purposes of the present invention, transient expression systems are useful e.g. for identifying SOX1 expressing cells or for inducing a pluripotent cell to differentiate.

Construction of vectors according to the invention employs conventional techniques, for example as described in Sambrook et al., 1989. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to generate the plasmids required. If desired, analysis to confirm correct sequences in the constructed plasmids is performed in a known fashion. Suitable methods for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing analyses for assessing gene expression and function are known to those skilled in the art. Gene presence, amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA, dot blotting (DNA or RNA analysis), or in situ hybridisation, using an appropriately labelled probe which may be based on a sequence provided herein. Those skilled in the art will readily envisage how these methods may be modified, if desired.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.

"comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

The invention is described, for the purpose of illustration only, in the following examples.

MATERIAL AND METHODS 25 Manufacture of SOX1 polyclonal antibodies: A 622bp HincI fragment encoding sequences C-terminal of the HMG box of SOX1 (207 is fused in frame to the bacterial GST gene in the construct pGEX3X. Fusion protein is induced and purified as described by Smith and Johnson (1988) Gene 67:31rabbits are treated with a course of injections as recommended by Smith and Johnson (1988): each injection contains 250pg of fusion protein. Two final bleeds, FB43 and FB44, are obtained from the rabbits prior to the preparation of polyclonal sera.

Immunocytochemistry: Embryos, P19 cells and neural plate explants are examined using standard techniques (Placzek et al., (1993) Development 117:205-218). Antibodies are used at the following dilutions: anti-SOX1 PAb (1:500); K2 anti-HNF3 MAb (1:40) 6G3 anti-FP3 MAb anti-3A10 MAb anti-2H3 (Neurofilament-160) MAb 4D5 anti-Islet-1 MAb (1:1000); anti-SSEA1 MAb (1:80) (Hybridoma Bank); anti-NESTINE MAb (1:10) (Hybridoma Bank); anti-BrDU MAb (1:500) (Sigma); Appropriate secondary antibodies (TAGO and Sigma) are conjugated to fluorescein isothiocyanate (FITC), Cy2 or Cy3.

BrDU analysis: Pregnant mice are injected intraperitoneally with 50ug/g of body weight of 5-bromo-2deoxyuridine (BrDU) (Sigma) in 09.% NaCl and sacrificed two hours after injection. Embryos are fisxed and sectioned as described above. The slides are washed twice in PBS, and incubated in 0.2% 15 HCI at 37 0 °C for 30 minutes, then rinsed thoroughly with PBS, followed by three rinses with PBS/0.1% *o.oo.

**o *o• *o• WO 99/00516 PCT/GB98/01862 31 Trinton/l% heat inactivated goat serum Mlonoclonal anti-BrDU (1:500 dilution in P-T-G) is applied to the sections and incubated at 4°C overnight.

Sequential sections are incubated in SOX1 antibody (1:500 dilution in P-T-G) at 4°C overnight. The slides are washed twice in P-T-G, then incubated in the appropriate secondary antibody for 30 minutes at room temperature, washed with P- T-G and mounted.

P19 cell cultured and retinoic acid treatment: P19 cells are cultured as previously described (Rudnichy and McBurney, 1987). To induce differentiation, cells are allowed to aggregate in bacterial grade petri dishes alone, in the presence of 1.M retinoic acid or in the presence of 1pM retinoic acid or in the presence of IPTG. After 4 days of aggregation in the presence of inducing agents, cells are plated on tissue culture chamber slides. The cells are allowed to adhere and grow for 4-5 days, with media changes every 24 hours. For immunoflurescence, cells are grown on tissue culture chamber slides coated with 0.1% gelatin, washed once with PBS, fixed at room temperature in Ix MEMFA for 1 hour, washed in P-T-G twice; then stained with appropriate antibody.

Cell counting analysis: For cell counting experiments P19 transfectant cell lines are induced to differentiate, plated on gelatine coated slides, fixed at room temperature in 1xMEMFA for one hour at day 6-8 for neurons. Cells are stained with Neurofilament (2H3) antibody and photographed using an Olympus fluorescence microscope. Cell counts are expressed as percentages of total cells inf a field. Eight fields from two different experiments are counted for each P19 clone.

Plasmids and transfection: To construct the SOX1 expression vector, pRSVopSoxl, the POP113CAT operator vector (Stratagene) is digested with Notl, end-filled Kpn/Stu (position 431-1694) fragment of the Soxl cDNA. The P3'SS, eukaryotic Lac repressor expressing vector (obtained from Stratagene) is transfected into P19 cells by lipofection. Stable transformants are selected in 250 p.g/ml of hygromycin. Expanded clones (250) are isolated and examined for expression of WO 99/00516 PCT/GB98/01862 32 the Lac repressor by indirect immunofluorescence with anti-lac PAb (Stratagene).

Four cell lines are isolated (P3'SS-10, 13, 22 and 47) which show ubiquitous and constitutive expression of the Lac repressor. P3'SS-10 is chosen for the subsequent experiments. P3'SS-10 is then transfected with pRSVopSoxl by lipofection. Stable clones are selected using 500p.g/ml G481. 250 clones are expanded and analysed for inducible Soxl expression by RNase protection and immunocytochemistry with SOX1 antibody.

RNase protection assays: Total RNA is prepared from P19 cells and RNase 0o protection assays are carried out using 5pg of P19 cell RAN as described by Capel et al., (1993) Cell 73:1019-1030. Anti-sense labelled probes are derived from the 396 bp SmaI-BspH1 fragment (position 1467-1863) of the Soxl cDNA, a 215bp Bsal exon 4 specific fragment of Wntl cDNA, a PvuII digest of the Mashl cDNA (Johnson et al., (1992) Development 114:75-87) and a NotI digest of SAP D cDNA is used a loading control (Dresser et al., (1995) Hum. Mol. Genet. 4:1613-1618).

RT-PCR: Total RNA is prepared from P19 cells as described by Capel et al., (1993). Reserve transcription, PCR reaction, and priming is performed as described by Okabe et al., (1996).

Rat lateral neural plate explants: Lateral neural plates (LNP) are isolated from days 8.5-9.0 rat embryos from prospective hindbrain and spinal cord regions as previously described (Placzek et al., 1993). Notochord explants are dissected fromi HH stage 608 chick embryos as previously described (Placzek et al., 1993).

Explants are embedded in collagen and cultured (Placzek et al., 1993) for 24, 48 and 96 hours. Purified rat SHH-N (Ericson et al., (1996) Cell 87:661-673) is added to cultures at concentrations within the effective ranges used in other assays (Ericson et al., 1996) WO 99/00516 PCT/GB98/01862 33 EXAMPLE 1 SOX1 IS EXPRESSED DURING EARLY NEURAL DEVELOPMENT SOX1 expression during mouse and rat neurulation is analysed using a rabbit polyclonal antibody against the SOX1 C-terminal region. In the mouse, expression of SOX1 is first detected at 7.5 days post coitum (dpc) in the anterior half of the late-streak egg cylinder. Cross-sections through the embryo at this stage reveal expression in columnar ectodermal cells, which appear to define the neural plate, while cells located more laterally are negative. Thus, SOX1 expression at this stage 0o is specific to the neural plate. SOX1 is maintained in all neuroepitheial cells along the entire anteroposterior axis as the neural pate bends (8.0-8.5 dpc, as shown in cross-sections of a 2 somite mouse embryos where Soxl expression is limited to neural folds) and fuses to form the neural tube (9.0-9.5 dpc, where Soxl labelling is seen to be restricted to the neural tube in cross-sections of 10-12 somite mouse embryos). The pattern of expression of SOX1 in the rat is similar to that in the mouse. The expression of SOX1 throughout the neural plate and early neural tube implies a similarity amongst these cells.

After neural tube closure, neuroepithelial cells begin to differentiate into defined classes of neurons at specific dorsoventral positions within the spinal cord (Altman and Bayer (1984) Adv. Anat. Embryol. Cell Biol. 85:32-46; Tanabe and Jessell, (1996) Science 274:1115-1123). As development proceeds, Soxl is downregulated in a stereotyped manner in cells alone D/V axis of the neural tube.

In the spinal cord, expressions first downregulated in cells that occupy the ventral midline (cross-sections of the thoracic region of 20 somite mouse embryos reveal a lack of SOX1 staining in this area), then the ventral motor horns (corresponding lack of staining being visible in cross section of 30-35 somite embryos) and subsequently the dorsal regions. These regions appear to correlate with floor plate, motor neurons and sensory relay interneurons, respectively.

WO 99/00516 PCT/GB98/01862 34 To ascertain this a series of antibody double-labelling experiments are performed in rat embryos. The SOX1 antibody is used in combination with a panel of antigenic markers which identify cells of the floor plate and mature neurons (Neurofilament labelled with contrasting colour markers and visualised in an Ell rat embryo). Expression of SOX1 and expression of these markers is almost entirely mutually exclusive. In the ventral spinal cord or the 10.0-12.0 dpc mouse embryo, SOX1 expression is maintained only in 'region X' (Yamada et al., (1991) Cell 64:635-647), as revealed by immunolabelling of two streams of cells located between the differentiated floor plate and ventral motor horns in 30-35 somite embryos. Eventually, by 13.5 dpc, SOX1 expression is restricted to a thin ventricular zone in the CNS. SOX1 expression in to detected in the peripheral nervous system (PNS). These expression profiles suggest that SOX1 is expressed by early neural cells in the CNS and is downregulated in the developing neural tube coincident with neural differentiation.

EXAMPLE 2 SOX1 MARKS PROLIFERATION CELLS WITHIN THE EMBRYONIC NEURAL TUBE The uniform expression of SOX1 in the neural plate and early neural tube followed by its down regulation along the D/V axis and restriction to the ventricular zone is reminiscent of the pattern of cell proliferation in the developing central nervous system (Sauer, (1935) J. Comp. Neurol. 62:377-405; Fujita, (1963) J. Comp.

Neurol. 120: 37-42; Altman and Bayer, 1984). In the neural plate and early neural tube, proliferating progenitor cells are organised in a pseudostratified epithelium in which the processes of these cells extend from the inner luminal to the outer mantle surface. At later stages the neural tube becomes progressively thicker and can be divided into different zones. The proliferating CNS progenitors are largely restricted to the inner ventricular zone (VZ) around the lumen. They begin to migrate away from the lumen while in S-phase, and after completing their final WO 99/00516 PCT/GB98/01862 mitosis, migrate to the outer layer, the marginal zone In the 10.5 dpc mouse embryo, SOX1 expression is detected, using an anti-SOX1 antibody, throughout the pseudostratified epithelium of the posterior neural tube and is restricted to the ventricular zone in more mature anterior region of the neural tube. In order to evaluate the relationship between SOX1 expression and proliferating CNS cells are directly assayed proliferation by monitoring the incorporation of bromodeoxyuridine (BrDU) with an anti-BrDU antibody. Pregnant mouse females at 10.5 dpc are injected with BrDU two hours prior to dissection to detect proliferating cells.

Embryos are then fixed, sectioned and double-labelled for BrDU incorporation and SOX1 expression. Similar to SOX1 expressing cells, those that incorporate BrDU are found throughout the posterior neural tube in 10.5 dpc mouse embryos and lie in the ventricular zone of the anterior neural tube. All cells that incorporate BrDU also express SOX1. SOX1-positive cells that do not incorporate BrDU are restricted to the luminar surface of the ventricular zone. In contrast, no SOX1 nor BrDU-positive cells are detected in the outer marginal zone. These results show that SOX1 is expressed in dividing neuroepithelial cells within the embryonic CNS.

EXAMPLE 3 SOX1 IS DOWNREGULATED IN COMMITED CELLS The mutual exclusion of SOX1 and markers of committed differentiated cells such as Isletl (Pfaff et al., (1996) Cell 84:1-20) raises the possibility that the downregulation of SOX1 may be a pre-requisite step for the differentiation in neural plate explants in vitro. Isolated neural plates explants are cultured with known inducers of ventral neural cells, namely the notochord and purified Sonic Hedgehog protein. The expression of SOX1 and incorporation of BrDU is then compared to the expression of three markers of ventral cells, Isletl, FP3 and HNF3P.

Consistent with our observations in vivo both the expression of SOX1 and Isletl as well as SOX1 and FP3 is mutually exclusive in neural plate explants cultured adjacent to notochord 8) or in the presence of purified Sonic Hedgehog protein as seen in E9 rat neural plate tissue cultured with Sonic Hedgehog protein for 48 WO 99/00516 PCT/GB98/01862 36 hours and stained with anti-SOX1 and anti-Isletl antibodies. Similarly, the incorporation of both BrDU and Isletl as well as BrDU and FP3 (detected using an anti-FP3 antibody) is mutually exclusive. In contrast, the domain of expression of HNF3P is found to extend beyond that of FP3 and into the region of BrDU positive cells.

To determine whether a similar population of cells could be detected in vivo embryos are analysed, and for co-expression of FP3 and HNF3p and for coexpression of BrDU and HNF3p. We find that medial floor plate cells co-express HNF3P and FP3 but do not incorporate BrDU, whereas lateral floor plate cells express only HNF3P and incorporate BrDU. HNF3P thus provides a marker for cells that are mitotically active but have begun to differentiate.

These cells, occupying the medial regions of the floor plate, express HNF3p but not SOX1. In contrast cells occupying lateral regions of the floor plate co-express HNF3p and SOX1. These observations, together with the mutually exclusive expression of SOX1 with Islet 1 and FP3 in ventral neural cells provide evidence that SOX1 is downregulated as cells exit mitosis and not at the onset of cell differentiation.

EXAMPLE 4 SOX1 EXPRESSION IS ASSOCIATED WITH NEURAL DIFFERENTIATION Neural induction is accompanied by the onset of new gene expression which in turn enables the formation of neural rather than epidermal tissue. The early and apparently uniform expression of SOX1 in neural cells, together with observations that Sox genes may affect cell lineage decisions, raises the possibility that SOX1 expression is an early response to neural inducing signals and that its expression may be involved in directing cells towards a neural fate. To address whether SOX1 plays a role in establishing neural fate in response to A P19 cell culture system is WO 99/00516 PCT/GB98/01862 37 used as an in vivo model system in which to analyse SOX1 expression and the effects of its misexpression.

P19 cells are an embryonal carcinoma cell line with the ability to differentiate into all three germ layers (McBurney, (1993) Int. J. dev. Biol. 37:135-140). In the undifferentiated state P19 cells morphologically resemble an uncommitted primitive ectodermal cell and express the cell surface antigen SSEA-1. These cells have a very low rate of spontaneous differentiation when grown in a monolayer in the absence of chemical inducers. P19 cells grown as aggregates, however, differentiate partially into endodermal cells. Furthermore, with the addition of retinoic acid, aggregated P19 cells differentiate into neuroepithelial-like cells (Jone- Villeneuve et al., (1982) J. Cell. Biol. 94:253-262). These express neuroepithelial markers such as NCAM, intermediate filament NESRIN, MASHI (Johnson et al., 1992) and WNT1 (St. Arnaud et al., (1989) Oncogene 4:1077-1080). When plated onto a substrate, about 15% of these cells differentiate into mature neurons expressing Neurofilament. Thus, in this in vitro model system retinoic acid acts as a "neural inducer".

Initially, the expression of Soxi in P19 cells is examined by both RNase protection and immunocytochemistry. The features of Soxl expression in P19 cells are similar to those observed in prospective neural tissue in vivo. Soxl mRNA and protein can not be detected in undifferentiated P19 cells which express the cell-surface antigen SSEA1 when analysed using anti-SOX1 and anti-SSEA antibodies, and by RNase protection. Similarly, when P19 cells are differentiated as aggregates without the addition of chemical inducers, SOX1 is not expressed as determined by RNase protection. In contrast, SOX1 is rapidly induced during neural differentiation when aggregated P19 cells are differentiated in the presence of retinoic acid. Soxl thus behaves similarly to other neuroepithelial markers such as Mash 1 and Wnt 1, the transcripts of which are detected in retinoic acid-treated P19 cells by RNase protection.

WO 99/00516 PCT/GB98/01862 38 When retinoic acid-treated P19 cell aggregates are plated onto tissue culture substrate, about 15% of the cells differentiate into mature process-bearing, Neurofilament-expressing neurons. Double-label immunofluorescence is used to simultaneously detect SOX1 and Neurofilament, to examine the expression of SOX1 in P19 cells displaying a fully differentiated neuronal morphology. SOX1 immunoreactivity is not detected in process-bearing Neurofilament-positive neurons.

Thus, as in vivo, SOX1 is expressed by P19 cells when they first assume a neural fate but it is then downregulated with their differentiation.

EXAMPLE USE OF SOX1 TO DIRECT CELLS TO A NEURAL FATE The previous data suggest that in P19 cells, as in vivo, SOX1 expression is induced at a time when neuroepithelial cells begin to differentiate. If SOX1 plays a role in directing cells towards the neural fate, expression of SOX1 in P19 cells may be able to substitute for retinoic acid to initiate neural differentiation. Endogenous SOX1 is accordingly activated in P19 cells using an inducible eukaryotic lac repressoroperator expression system. To establish this system a clonal line of P19 cells is generated which constitutively and ubiquitously expresses the lac repressor. This parent line (P3'SS-10) is transfected with pRSVopSoxl, a vector containing the Soxl cDNA under the regulation of an inducible RSV promoter and stable lines are established. In the uninduced state, without the addition of isopropyl-P-dthiogalactase (IPTG) these lines express high levels of the lac repressor that binds to operon sites upstream of the RSV promoter and thus blocks transcription of Soxl.

Upon addition of IPTG a conformational change occurs, decreasing the affinity of the repressor and resulting in the activation of pRSVopSoxl. Approximately 250 clones of transfectants are isolated in the repressed state. Using RNase protection and immunocytochemistry assays three clones are selected (708-13, 708-16 and 708- 21) that express high levels of RSVopSoxl in response to IPTG.

WO 99/00516 PCT/GB98/01862 39 The pluripotentiality of these clones is not compromised by the transfection and selection. All three lines express SSEA1 in the uninduced state. Furthermore, when aggregated in retinoic acid the uninduced clones initiate expression of endogenous Soxl and differentiate into mature Neurofilament-expressing neurons after plating, in a manner similar to wild-type P19 untransfected cells.

In order to address whether expression of SOX1 can initiate neural differentiation and thereby substitute for the requirement of retinoic acid, it is determined whether the transient exposure of P19 aggregates to retinoic acid can be replaced by a transient induction of RSVopSoxl, through addition of IPTG. Wildtype P19 cells and transfected P19 clones (708-13, 708-16 and 708-21) are cultured as aggregates for 96 hours with or without the addition of IPTG. After 96 hours RNA is isolated from half of the aggregates for RNase protection and/or RT-PCR assays. The remaining aggregates are plated onto tissue culture substrate, allowed to differentiate for three days without further addition of IPTG and then scored for the expression of a panel of neuroepithelial and neuronal markers by immunocytochemistry. These conditions are the same as those used for retinoic acid-induced differentiation of wildtype P19 cells. After 96 hours the clones induced to express RSVopSoxl with IPTG express endogenous Soxl and Mashl.

The expression of these two neuroepithelial markers is similar to that seen in wildtype cells induced with retinoic acid. In addition the IPTG induced clones expressed NESTIN and Hoxa7 (Mahon et al., (1988) Development (Suppl.) 187- 195). Further differentiation of the transiently-induced clones on substrate showed the presence of mature neurons as demonstrated by Neurofilament-positive, 3A10positive and Isletl-positive cells. All three clones 708-13, 708-16 and 708-21 differentiate in this matter although the number of mature neurons produced is variable. The number of differentiated neurons formed in the IPTG induced clones is estimated by determining the number of Neurofilament-positive cells in a given field of cells. The number of neurons ranges from 6-8% for clone 708-13, 15-20% for clone 708-16 and 20-25% for clone 708-21. The latter two clones show uniform and ubiquitous induction of SOX1 expression whereas expression in clone 708-13 is WO 99/00516 PCT/GB98/01862 not in all cells. In addition, the transiently induced clones generate GFAP-positive cells indicating glial cell differentiation. None of these markers is detected in wildtype P19 cells cultured in the presence of IPTG or in clones 708-13, 708-16, and 708-21 cultured in the absence of IPTG. The expression of SOX1, both in vivo and in vitro, is mutually exclusive with mature neuronal markers such as Neurofilament and Isletl. To examine SOX1 expression in the mature neurons generated in the transiently-induced clones, double-label immunoflourescence is used to simultaneously detect SOX1 and Neurofilament. No SOX1 expression could be detected in cells positive for Neurofilament in these cultures.

WO 99/00516 PCT/GBRq98/01862 41 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: MEDICAL RESEARCH COUNCIL STREET: 20 PARK CRESCENT CITY: LONDON COUNTRY: UK POSTAL CODE (ZIP): W1N 4AL (ii) TITLE OF INVENTION: NEURONAL STEM CELL GENE (iii) NUMBER OF SEQUENCES: 6 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (EPO) INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 2312 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Gallus gallus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CCGCAGAGCG CGGCAGGACG GGCACACGCC GGGCGCAGCA CCGCGCAGCC CCGCACCTGT TTGCGCGCCC CGCGCCCGGA GACGCCGGCA GCGCCGCCGC CGCCGCTCCT CGCATGTGCG GCCGGAGGGA AGTGAGGAAG CCCCGTGAAT GTACAGCATG CTCGCCCGGC GGAGCCCCGG CGCCCGGCGG CGGCCTCTCG CGGCGGCGGC GGCGGCGGCG GCGGCGGGGG CAAAGCGGGG

CCGCGAAGCA

GCGGCCCCCG

GTGCCTCCCC

ATGATGGAGA

GGGCAGAGCG

CAGGACCGCG

CCGCGCAGCC

GCAGCGGGAG

GCCGCCCGGC

CGGACTTGCA

GCGCGGGCGG

TGAAGCGCCC

120 180 240 300 360 WO 99/00516PC/B/086 PCT/GB98/01862

CATGAACGCC

CAAGATGCAC

GGCCGAGAAG

GCACCCGGAT

GTACTCGCTG

CGGCGTGGGC

CGGCGGGGCG

GGGCTCGGTG

CGGGCAGCAC

CCCGCACAAC

CTCCAACTCG

CTCGCAGCCC

CTCGTCGGGC

GCCCGTCACC

GTACTTACCG

CTCCCTGCCC

GCACATCTGA

C-GCAGGACTG

ATTCGGCTCC

TTTTCTTTTC

TAATAATAAT

TTGTTTTTAG

AGACTTTTTG

AGGAGAGAGA

ACGAA.AAGAC

TGCGCTTCGC

TTCCTCGCAC

TTTGGTTGGG

TTCATGGTGT

AACTCGGAGA

CGGCCTTTCA

TATAAATACC

GCCGGAGGGC

ATGGGCGTCG

GCGGGCGGCG

GCGGCGGCGG

CCGGGCGGCG

CCGCAGCCCA

CAGGGCTACG

CACCAGAACT

GCGCTGGGCG

TCGCACTCGC

GGCGGCGAGG

CAGCACTACC

GCGGCCCCGG

CGGCCCCGCC

TTCCCGCCCT

TTCCTTTTTG

AATAACGAGA

GAGGACCAGA

TACAGTATTT

GAGGATTAAA

AAAACCATCG

CCCGCACCGC

ACCCCAAAAC

TTATTTCGGC

GGTCGCGGGG

TCAGCAAGCG

TCGACGAGGC

GGCCCCGGCG

TGCTGGGCGC

GCGTGATCCC

GCTACGCGCA

CGGCGGCGGC

GGGGGCACCC

TGCACCGCTA

TGAGC GC CT C

CGGCGGCCGC

CGCTGGGCTC

GGGCCCCGTG

GAGGCGACCC

AGAGCGCCAG

AGCGGCCCCG

GCCGCCCCGC

CCCCCTCCCT

TACAGAAATG

GAGAGAGAAA

TCCCGGGACT

ATCATTCACC

AAACAAAAAC

CCGCTGACAC

CTCACTGCAA

AGCACCACGA

TGCTTTATTT

GCAGCGGCGG

GCTGGGCGCC

GAAkGCGGCTG

GAAGACCAAG

CGGCCCGGCC

CGGCGGAGTC

CATGAACGGG

GGCGATGATG

GCACCACCCG

CGACATGGGC

GCCCTCGGGC

GGCGGCGGCG

GCTCGTCAAG

CCCCGGGGAC

GGCGGCCGCC

CACGGGGGTC

GAGCGGCGCG

GCCCGCCGCC

CCTTCCTTTT

TTTTGATGTT

AAGAAGGTAA

AGTTTTAGAC

CCAGAGACAC

AAACAAACAA

CCAAAGTTCG

ACGGAGCCGA

GTTTCCGTAG

ATACAACTTT

AAGATGGCCC

GAGTGGAAGG

CGGGCGCTGC

ACGCTGCTCA

GCGGGCGGCC

GGGCAGCGGC

TGGGCCAACG

CAGGAGGCGC

CACCCGCACC

GCGCTGCAGT

TACGGCGCGC

GCGGCGGCGG

TCGGAGCCCA

CTGCGGGAGA

GCCGCCCAGA

AACGGCACCG

GAGGGCCCCG

CCCCTTCGTT

TTTGTTTTGT

CTTGTAATAA

CGGTGGCTTT

TGAACTTCTG

AGAGCGTTTA

ACAAACAAAA

GGCGGGGCCA

CGGGGAGCGG

ATGTTCTCGC

TTCTTCTTCT

AGGAGAATCC

TGATGTCGGA

ACATGAAGGA

AGAAGGACAA

CTCCCGCCGT

TGGAGAGCCC

GCGCCTACCC

AGCTCGCCTA

ACCCGCACCA

ACAGCCCCAT

TGCCCTACGG

CGGCGGCCGC

GCGTGAGCCC

TGATCAGTAT

GCCGCCTGCA

TCCCCTTGAC

GCCCGGGCCC

TTTGCCTTTC

TTTGTTTTGT

TAATAAATAA

ACTGACCTTT

TGTTTTATCG

TTTGCAAAAG

AAAGACGGCG

ACTTTCGGGC

TGCTCGTTCC

GCTTTTCCTT

TCCTTTCTTC

420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 WO 99/00516 WO 9900516PCT/GB98/0 1862 43 CCGAGGTTGC AACGTTTGCT TGATTTTTAT TTTATTTTAT TTTTTTTTCT GGGTTATGTG AAACTTTACT GTATCTGCAT CATTTCGGTT TGTTTTCCTC CCCCCCCCCC CCTTTTTTTT TTTTTTTACA TTTTTTTGTA TCATCTCGTG TAAATGCATT GTGAAATAAT TTTTATCTAG GCGTGGCGAG GGAACCCAGA CTGTACATAG TTTACTAAAA AGCCTTTCTG CTAAACAGAA ACCCGAAGGA TGCGTTCCAT TTTGAGTTAA AT INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 2376 base p~airs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Mus musculus 2100 2160 2220 2280 2312 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: ACAGGAACGG AGACTTCGAG CCGAGAAGAG GAGGCAGCGA ACCCTGCGTC GGGCCCAGGG

GCACCGCTTC

AGTTCAGCCC

TGGTTTGTGC

CTTTTGCCTC

CTAGGCACCC

CCCACCCACC

CCGCTA.GCCG

GCCCAGGCGC

GGGGGCGGCG

AACGCCTTCA

ATGCACAACT

GAGAAGCGGC

AGACCCAGAA

TGAGTGACCG

ACCGGGAGAA

TTTTTTGTTG

ACCGCACACA

CCCACCCGGC

CCCAGATGTA

CCACGAACCT

GCGGCGGCGG

TGGTGTGGTC

CGGAGATCAG

CGTTCATCGA

AGTGGAGCCT

GACGGCAGCA

ACTTTCCACC

TTGTTGTTGT

CACCCCCCCC

CGTCTATGCT

CAGCATGATG

CTCGGGCCCG

GGGCACCAAG

CCGCGGACAG

CAAGCGCCTC

CGAGGCCAAG

CAACTTGGCC

ATCAACCTGG

TGCGAGCTGG

TGTGGCCTCC

CCCAGTCTCT

CCAGGCCCTC

ATGGAGACCG

GCCGGGGCGC

GCCAACCAGG

CGGCGCAAGA

GGGGCCGAGT

AGACTGCGCG

ACGACTGCAC

CCATCGGCCT

ACCCGCGCTA

ACCCAACCCC

CTGGGCTGAT

TCTTTGCGGT

ACCTGCACTC

GCGGGGGCGG

ATCGGGTCAA

TGGCCCAGGA

GGAAGGTCAT

CGCTGCACAT

CTGTTTGCAC

CTTTGGCAAG

AGTGCGTGTG

CTTCTCTCCG

CCTCTCTCCA

ACCGGTGAAC

GCCCGGCGGC

CGGTGGGGGC

GCGGCCCATG

AAACCCCAAG

GTCCGAGGCC

GAAGGAACAC

120 180 240 300 360 420 480 540 600 660 720 780 WO 99/00516PC/B8086 PCT/GB98/01862 44 CCGGATTACA AGTACCGGCC GCGCCGCAAG ACCAAGACGC TGCTCAAGAA GGACAAGTAC

TCGCTGGCCG

GGTGTGGGCG

GCGGCGGGAG

GTGGCCGCGG

CACCCAGGCG

CCGCACGCGC

TACAGCCCCA

ATCCCTTACG

GCGGTGGCGG

TCTCTGGTCA

TGTCCCGGGG

CCGGCGGCGG

CAGGGCGCGG

GACGCCGGGG

CCCAGCCTCC

TGGAGAGCCG

TGACACCAGA

ATTTATCGTC

AACCCAGCGA

TTACTTCCCG

GTTATAAATC

CTTTTGGGTT

TGGGTTTTGT

TCTTGTAAAT

CGGATGTTGT

GCGGGCTGCT

TGGGCGTCGG

GAGGCTACGC

CGGCTGCGGC

CGGGCGGCCG

ATCCTCACAA

TCTCCAACTC

GCGCCGCGGC

CAGCGGCAGC

AGTCGGAGCC

ACCTGCGCGA

CAGCGGCTGC

GCGCGGGCGT

ACACTGCGGC

AGAGCCCGAC

AGCGCCAGGT

CTGGCCTCTT

CGCAGAGGAG

GGCGATGCCA

CCAGCTCTTC

TAAGTAAGGC

GGTTGGTTAA.

AAATCTCTGT

GCGTTGTGAC

ACATAGCCTG

AGCGGCCGGC

GGCGGCGGCG

GCATGTCAAC

CGCGGCCATG

GCACCCGCAC

CCCGCAGCCC

TCAGGGCTAC

CGCCGCCGCC

CGCGGCAGCC

GAGCGGCAGT

GATGATCAGC

GGCGGCCCAA

CAACGGCACG

TTAAGGCCGG

TTTGTACCGA

GACATCTGCC

AGACTGAACT

GCACACAGCG

ACTTTTGTAT

TGCCTGAGGC

AGATCCAAAC

TTTATACCGC

ATCCGAGCAT

ATTTTTATTT

CAAGTCTTTC

GCGGGTGGCG

GTGGGCCAGC

GGCTGGGCTA

ATGCAGGAGG

GCACACCCGG

ATGCACCGCT

ATGAGCGCGT

GCCGCTGCGG

GCGTCGTCGG

CCGCCGGCCC

ATGTACCTGC

AGCCGGCTGC

GTGCCCCTGA

CGCCCCGGCG

GGTCCCCGCG

CCCATCACCT

TTGGTGTTTT

TTTTCTCGGC

GACTGCCGGC

CGAGTGACGG

ATTTACAAGT

AATCCCCTCT

TTCCAATTTT

TAGGCGTTGC

ATCTAAAAGC

GCGGCGCGGC

GCTTGGAGAG

ACGGCGCCTA

CACAGCTGGC

CGCACCCGCA

ACGACATGGG

CGCCTTCGGG

GCGGCGCGCA

GGGCCCTGGG

CGGCTCACTC

CGGCCGGCGA

ACTCGCTGCC

CGCACATCTA

ACGAAGAGCG

CTCTCGATAA

TCCCCAGGAC

CATGAGACCT

TTCGGAGGAC

TCTGTAACTT

ACCTCGAGCC

TTTTTGTAGT

CAGACGGTGG

TTGTTTTGTT

GATACGGGGG

CGTGGCCATG

CCCAGGCGGC

CCCCGGCTCG

CTACGGGCAG

CCCGCACCAT

CGCGCTGCAG

CTACGGCGGC

CCAGAACTCG

CGCCCTCGGA

ACGGGCACCG

GGGTGGCGAC

ACAGCACTAC

GCGCCGCGGG

AGGCCTGCGC

AAGGCCGCTC

TCCGAGGCGC

TTTGTACAGT

AAAAGACAAA

TTTCCGGGGT

CTTCTCACTT

TGTTACCGCT

AGTTATATTC

TTGTATTATT

GAAGAGGAGT

900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2376 AAAAACA-AAG AGAGATACCC TAAGAG CCAAAATGCA TCAAATTTGA ACAATACATT INFORMATION FOR SEQ ID NO: 3: WO 99/00516 PCT/GB98/01862 SEQUENCE CHARACTERISTICS: LENGTH: 1542 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (ix) FEATURE: NAME/KEY: CDS LOCATION:60..1223 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: CCGGCCGTCT ATCTCCAGGC CCTCTCCTCG CGGTGCCGGT GAACCCGCCA GCCGCCCCG ATG TAC AGC ATG ATG ATG Met Tyr Ser Met Met Met 1 5 CAG GCC CCC ACG AAC CTC Gin Ala Pro Thr Asn Leu GAG ACC GAC CTG CAC TCG Glu Thr Asp Leu His Ser 10 TCG GGC CCC GCC GGG GCG Ser Gly Pro Ala Gly Ala 25 CCC GGC GGC GCC Pro Gly Gly Ala GGC GGC GGC GGG Gly Gly Gly Gly AAG GCC AAC CAG Lys Ala Asn Gin TGG TCC CGC GGG Trp Ser Arg Gly GGC GGA GGC Gly Gly Gly GGG GGC GGC Gly Gly Gly GGC GGC Gly Gly 40 GGC GGG GGC GCC Gly Gly Gly Ala GAC CGG Asp Arg GTC AAA CGG CCC Val Lys Arg Pro

ATG

Met AAC GCC TTC ATG Asn Ala Phe Met 251

CAG

Gin CGG CGC AAG ATG Arg Arg Lys Met CAG GAG AAC CCC Gin Glu Asn Pro ATG CAC AAC TCG Met His Asn Ser ATC AGC AAG Ile Ser Lys AAG CGG CCG Lys Arg Pro CGC CTG Arg Leu GGG GCC GAG TGG Gly Ala Glu Trp GTC ATG TCC GAG Val Met Ser Glu GCC GAG Ala Glu

TTC

Phe 100 ATC GAC GAG GCC AAG CGG CTG CGC GCG Ile Asp Glu Ala Lys Arg Leu Arg Ala 105 CTG CAC ATG Leu His Met 110 395 AAG GAG CAC CCG GAT TAC AAG TAC CGG CCG CGC CGC Lys Glu His Pro Asp Tyr Lys Tyr Arg Pro Arg Arg 115 120 AAG ACC AAG ACG Lys Thr Lys Thr WO 99/00516 PCT/GB98/01862 CTG CTC AAG Leu Leu Lys 130 AAG GAC AAG TAC TCG CTG GCC GGC Lys Asp Lys Tyr Ser Leu Ala Gly

GGG

Gly 140 CTC CTG GCG GCC Leu Leu Ala Ala

GGC

Gly 145 GCG GGT GGC GGC Ala Gly Gly Gly GGC GCG Gly Ala 150 GCT GTG GCC ATG Ala Val Ala Met 155 GGC GTG GGC GTG Gly Val Gly Val GTG GGC GCG GCG Val Gly Ala Ala GTG GGC CAG CGC Val Gly Gin Arg

CTG

Leu 170 GAG AGC CCA GGC Glu Ser Pro Gly GGC GCG Gly Ala 175 GCG GGC GGC Ala Gly Gly

GCG

Ala 180 TAC GCG CAC GTC Tyr Ala His Val GGC TGG GCC AAC Gly Trp Ala Asn GGC GCC TAC Gly Ala Tyr 190 ATG CAG GAG Met Gin Glu CCC GGC TCG GTG GCG GCC GCG GCG Pro Gly Ser Val Ala Ala Ala Ala 195 200 GCC GCC GCG GCC Ala Ala Ala Ala GCG CAG Ala Gln 210 CTG GCC TAC GGG Leu Ala Tyr Gly CAC CCC GGC GCG His Pro Gly Ala GGC GCG CAC CCG Gly Ala His Pro

CAC

His 225 CGC ACC CCG GCG Arg Thr Pro Ala CCG CAC CCG CAC Pro His Pro His

CAC

His 235 CCG CAC GCG CAC Pro His Ala His

CCG

Pro 240 779 CAC AAC CCG CAG His Asn Pro Gln ATG CAC CGC TAC Met His Arg Tyr

GAC

Asp 250 ATG GGC GCG CTG Met Gly Ala Leu CAG TAC Gin Tyr 255 AGC CCC ATC Ser Pro Ile TAC GGC GGC Tyr Gly Gly 275 AAC TCG CAG GGC Asn Ser Gin Gly

TAC

Tyr 265 ATG AGC GCG TCG Met Ser Ala Ser CCC TCG GGC Pro Ser Gly 270 GCC GCG CAC Ala Ala His CTC CCC TAC GGC Leu Pro Tyr Gly

GCC

Ala 280 GCG GCC GCC GCC Ala Ala Ala Ala

GCC

Ala 285 CAG AAC Gin Asn 290 TCG GCC GTG GCG Ser Ala Val Ala GCG GCG GCG GCG Ala Ala Ala Ala

GCG

Ala 300 GCC GCG TCG TCG Ala Ala Ser Ser

GGC

Gly 305 GCC CTG GGC GCG CTG GGC TCT CTG GTG Ala Leu Gly Ala Leu Gly Ser Leu Val

AAG

Lys 315 TCG GAG CCC AGC Ser Glu Pro Ser AGC CCG CCC GCC CCA GCG CAC TCG CGG Ser Pro Pro Ala Pro Ala His Ser Arg CCG TGC CCC GGG Pro Cys Pro Gly GAC CTG Asp Leu 335 1019 1067 1115 CGC GAG ATG ATC AGC ATG TAC TTG CCC GCC GGC GAG GGG GGC GAC Arg Glu Met Ile Ser Met Tyr Leu Pro Ala Gly Glu Gly Gly Asp 340 345 350

CCG

Pro WO 99/00516 PCT/GB98/01862 GCG GCG GCA GCA GCG GCC GCG GCG CAG AGC CGG C Ala Ala Ala Ala Ala Ala Ala Ala Gin Ser Arg L 355 360 CAG CAC TAC CAG GGC GCG GGC GCG GGC GTG AAC G Gin His Tyr Gln Gly Ala Gly Ala Gly Val Asn G 370 375 3 ACG CAC ATC TAG CGCCTTCGGG ACGCCGGGGA CTCTGCG Thr His Ile 385 AGCTCGCGGC CCGCGCCCGG CTCCCGCCCC GCCCCGGCGC G CGTTCCCACA TTCTTGTCAA AAGGAAAATA CTGGAGACGA A CCCACTCACC TTCCCCGGAG ACCCTGGCGA CCGCCGGGCG C GACTGAACTT CGGTGTTTTC TTGAGACTTT TGTACAGTAT GGAAGCGTTT TCTTTGCTCG AGGGACAAAA AATGCAAAA INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 388 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Met Tyr Ser Met Met Met Glu Thr Asp Leu His 1 5 10 Gin Ala Pro Thr Asn Leu Ser Gly Pro Ala Gly 25 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 40 Asp Arg Val Lys Arg Pro Met Asn Ala Phe Met 50 55 Gin Arg Arg Lys Met Ala Gin Glu Asn Pro Lys 70 75 Ile Ser Lys Arg Leu Gly Ala Glu Trp Lys Val 90 Lys Arg Pro Phe Ile Asp Glu Ala Lys Arg Leu 100 105 Lys Glu His Pro Asp Tyr Lys Tyr Arg Pro Arg TG CAC TCG CTG CCG eu His Ser Leu Pro 365 GC ACG GTG CCC CTG ly Thr Val Pro Leu GCG GCGACCCACG GCGTGGCTT TTGTATCAGA CGCCGGGTG ACGCGTGTCC TGACACCAG ACTTGGTTTA :TATCACCTA CGGAGGAAGC 1163 1211 1263 1323 1383 1443 1503 1542 Ser Ala Ala Val Met Met Arg Arg Pro Gly Lys Trp His Ser Ala Lys Gly Gly Ala Ser Asn Glu Leu 110 Thr Gly Gly Asn Arg Ser Ala His Lys Ala Gly Gin Gly Glu Glu Met Thr WO 99/00516 WO 9900516PCT/GB98/01862 LeuI Gly 145 Val Ala Pro( Ala His 225 His Ser Tyr Gin Gly 305 Ser Arg Ala Gin Thr 385 (2) eu kla ,ly Gly

G

1 y Gln 210 Arg Asn Pro Gly Asn 290 Al a Prc Glh AlE 115 Lys Gly Ala Gly Ser 195 Leu Thr Pro Ile Gly 275 Ser Leu Pro Met Ala 355 Lys Asp Lys Tyr Gly Al a Al a 180 Val Al a Pro Gin Ser 260 Leu Al a Gly Al a Ile 340 Ala Gly Pro 165 Tyr Ala.

Tyr Al a Pro 245 Asn Pro Val Al a Pro 325 Ser Ala G1y 150 Val Al a Al a Gly His 230 Met Ser Tyr Ala Leu 310 Al a Met Ala 135 Ala Gly His Ala Gin 215 Pro His Gin Gly Ala 295 Gly His Tyi Al~ 120 Ser Ala Gin Val Ala 200 His His Arg Gly *Ala 280 *Ala Ser Ser Leu 3. Ala 360 Ja 1 Drg Asn 185 Ala Pro Pro Tyr Tyr 265 Ala Ala Leu Arg Pro 345 Glr Aeu Ala Gly Gly Leu Ala Ala Ala Leu 170 Gly Ala.

Gly His Asp 250 Met Ala Ala Val Ala 330 3Ala ISer Ie t 155 G1u rrp Ala Ala His 235 Met Ser Ala Ala Lys 315 Pro Gly Arg 140 Gly Val Ser Pro Ala Asn Ala Met 205 Gly Gly 220 Pro His Gly Ala Ala Ser Ala Ala 285 Ala Ala 300 Ser Glu Cys Pro *Glu Gly *Leu His 365 Gly Gly Gly 190 Met Ala Al a Leu Pro 270 Ala Ala Pro Gly Gly 350 Ser Val1 Gly 175 Ala Gin His His Gin 255 Ser Al a Ser Ser Asp 335 Asp Leu Gly 160 Al a Tyr Glu Pro Pro 240 Tyr Gly His Ser Gly 320 Leu Pro Pro His Tyr Gin Gly Ala Gly Ala Gly Val Asn 370 375 His Ile INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: Gly 380 Thr Val Pro Leu WO 99/00516 WO 9900516PCT/GB98/0 1862 49 LENGTH: 1161 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: variation LOCATION:replace(7. "agy") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(28. .30, "cun') (ix) FEATURE: NAME/KEY: variation LOCATION:replace(34. .36, 'agy") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(64. .66, "cun") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(67. .69, "agy") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(148. .150, "cgn") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(157. .159, "cgn") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(184. .186, "agy") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(187. .189, 'cgn') (ix) FEATURE: NAME/KEY: variation LOCATION:replace(196. .198, 'cgn') (ix) FEATURE: NAME/KEY: variation LOCATION:replace(199. .201, 'cgn") WO 99/00516 WO 9900516PCT/GB98/01862 (ix) FEATURE: NAME/KEY: variation LOCATION:replace(235. .237, "agy") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(250. .252, "cgn") (ix) FEATURE: NAME/KEY: variation LOCATIQN:replace(244. .246, "agy') (ix) FEATURE: NAME/KEY: variation LOCATION:replace(253. .255, "cun') (ix) FEATURE: NAME/KEY: variation LOCATIQN:replace(277. .279, "agy") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(291. .294, "cgn') (ix) FEATURE: NAME/KEY: variation LOCATION:replace(316. .318, "cgn') (ix) FEATURE: NAME/KEY: variation LOCATION:replace(319. .321, 'cun") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(322. .324, "cgn') (ix) FEATURE: NAME/KEY: variation LOCATION:replace(328. .330, "cun") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(361. .363, "cgn") (ix) FEATURE: NAME/KEY: variation LOCATION: replace (367. .369, "cgn") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(370. .372, "cgn") (ix) FEATURE: NAME/KEY: variation LOCATION: replace (385. .387, "cun") WO 99/00516 PCT/GB98/01 862 51 (ix) FEATURE: NAME/KEY: variation LOCATION:replace(388. .390, 'cun") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(406. .408, 'agy") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(409. .411, "cun") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(421. .423, "cun") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(424. .426, "cun") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(505. .507, llcgn") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(508. .510, 'cun") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(513. .515, "agy") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(583. .585, "agy') (ix) FEATURE: NAME/KEY: variation LOCATION:replace(631. .632, "cun') (ix) FEATURE: NAME/KEY: variation LOCATION:replace(676. .678, "lcgn") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(742. .744, llcgnll) (ix) FEATURE: s0 NAME/KEY: variation LOCATION:replace(760. .762, "cun') (ix) FEATURE: NAME/KEY: variation LOCATION:replace(769. .771, "agy") WO 99/00516 WO 9900516PCT/GB98/OI 862 52 (ix) FEATURE: NAME/KEY: variation LOCATION:replace(778. .780, "agy") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(784. .786, "agy") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(799. .801, "agy") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(805. .807, "agy") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(81..813, "agy") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(836. .838, "cun") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(871. .873, "1agy"l) (ix) FEATURE: NAME/KEY: variation LOCATION: replace (907. .909, "agy") (ix) FEATURE: NAME/KEY: variation LOCATION: replace (91.0. .912, "agy") (ix) FEATURE: NAME/KEY: variation LOCATION: replace (919. .921, "cun") (ix) FEATURE: NAME/KEY: variation LOCATION: replace (928. .930, "cun") (ix) FEATURE: NAME/KEY: variation LOCATION: replace (934. .936, "agy") (ix) FEATURE: NAME/KEY: variation LOCATION: replace (937. .939, "cun") (ix) FEATURE: NAME/KEY: variation LOCATION: replace (946. .948, "agy") WO 99/00516 WO 9900516PCT/GB98/OI862 53 (ix) FEATURE: NAME/KEY: variation LOCATION:replace(955. .957, 'agy") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(961. .963, "agy") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(982. .984, "agy") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(985. .987, "cgn") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(1006. .1008, "cun") (ix) FEATURE: NAME/KEY: variation LOCATION:rep].ace(1009. .1011, "cgn") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(1021. .1023, "agy") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(1030. .1032, 'cun") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(1084. .1086, 'agy') (ix) FEATURE: NAME/KEY: variation LOCATION:replace(1087..1089, "cgn") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(1090. .1092, "cun") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(1096. .1098, "agy") (ix) FEATURE: NAME/KEY: variation LOCATION:replace(1099. .1101, "cun") (ix) FEATURE: NAME/KEY: variation 11; LOCATION:replace(1150. .1152, "cun") WO 99/00516 WO 9900516PCT/GB98/01862 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: AUGUAYUCNA UGAUGAUGGA RACNGAYEJUR CAYUCNCCNG GNGGNGCNCA

RGCNCCNACN

AAYUUPRUCNG GNCCNGCNGG NGCNGGNGGN GGNGGNGGNG GNGGNGGNGG

NGGNGGNGGN

GGNGGNGGNG, CNAARGCNAA YCARGAYAGR GUNAAPAGRC CNAUGAAYGC NUtJYAUGGUN UCGUCNAGRG GNCAP.AGRAG RAARAUGGCN CARGARAALYC CNAARAUGCA

YAAYUCNGAR

AUHUCNAARA GRUURGGNGC NGARUCGAAR GUNAUGUCNG ARGCNGARAA RAGRCCNtJUY AUHGAYGARG CNAAPAGRUU RAGRGCNUUP- CAYAUGAARG ARCAYCCNGA

YUAYAARUAY

AGRCCNAGPRA GRAAP.ACNAA RACNUURUUP- AARAARGAYA ARUAYUCNUU

RGCNGGNGGN

UTJRUURGCNG CNGGNGCNGG NGGNGGNGGN GCNGCNGUNG CNAUGGGNGU

NGGNGUNGGN

GUNGGNGCNG CNCCNGUNGG NCAP.AGRUUR GARUCNCCNG GNGGNGCNGC

NGGNGGNGCN

UAYGCNCAYG UNAAYGGNUC GGCNAAYGGN GCNUAYCCNG GNUCNGUNGC

NGCNGCNGCN

GCNGCNGCNG CNAUGAUGCA RGARGCNCAP- UURGCN UAYG GNCARCAYCC

NGGNGCNGGN

GGNGCNCAYC CNCAYAGRAC NCCNGCNCAY CCNCAYCCNC AYCAYCCNCA

YGCNCAYCCN

CAYAAYCCNC ARC CNAUGCA YAGRUAYGAY AUGGGNGCN U URCARUAYUC

NCCNAUHUCN

AAYtJCNCARG GNUAYAUGUC NGCNUCNCCN UCNGGN UAYG GNGGNUURCC

NUAYGGNGCN

GCNGCNGCNG CNGCNGCNGC NCAYCAPAAY UCNGCNGUNG CNGCNGCNGC

NGCNGCNGCN

GCNGCNUCNU CNGGNGCNUU RGGNGCNUUP. GGNUCN UUR G UNAARUCNGA RCCNtJCNGGN UCNCCNCCNG CNCCNGCNCA YUCNAGRGCN CCNUGYCCNG GNGAYUURAG

RGARAUGAUH

UCNAUGUAYU URCCNGCNGG NGARGGNGGN GAYCCNGCNG CNGCNGCNGC

NGCNGCNGCN

CARUCNAGRU URCAYUCNUU RCCNCARCAY UAYCARGGNG CNGGNGCNGG

NGUNAAYGGN

ACNGUNCCNU UPACNCAYAU

H

INFORMATION FOR SEQ ID NO: 6: SEQUENCE

CHARACTERISTICS:

LENGTH: 7, base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

120 160 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1161 WO 99/00516 PCT/GB98/01862 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

WWCAAWG

Claims (13)

1. A method for isolating one or more neuroblastic cells from a population of cells comprising the steps of: detecting the expression of the Soxl gene in the cell(s); and sorting the cell(s) to isolate those cells expressing the Soxl gene. 0o 2. A method according to claim 1, wherein the population of cells is derived from CNS tissue.

3. A method according to claim 1, wherein the population of cells is derived from a cell culture.

4. A method according to any preceding claim, wherein the expression of the Soxl gene is detected by nucleic acid hybridisation. A method according to any one of claims 1 up to 3, wherein the expression of the Soxl gene is detected by a binding of SOX1 to a detectable ligand.

6. A method according to claim 5, wherein the detectable ligand is a labelled immunoglobulin.

7. A method according to claim 5, wherein the detectable ligand is a labelled oligonucleotide complementary to Soxl mRNA.

8. A method according to any preceding claim, wherein the expression of the Soxl gene is detected by FACS analysis. WO 99/00516 PCT/GB98/01862

9. A method for isolating a neuroblastic cell from a population of cells, comprising the steps of: transfecting the population of cells with a genetic construct comprising a coding sequence encoding a detectable marker operatively linked to a Soxl control region; detecting the cells which express the selectable marker; and sorting the cells which express the selectable marker from the population of cells. A method for isolating a neuroblastic cell from a population of cells, comprising the steps of: transfecting the population of cells with a genetic construct comprising a coding sequence encoding a detectable marker operatively linked to a control sequence which is transactivatable by SOX1; detecting the cells which express the selectable marker; and sorting the cells which express the selectable marker from the population of cells.

11. A method according to claim 9 or claim 10, wherein the selectable marker is a fluorescent or luminescent polypeptide.

12. A method according to claim 9 or claim 10, wherein the selectable marker is a polypeptide detectable at the surface of the cell. W oo/00n1C A TDr/T' O /m OLi v j 17.. .1 I'.3D7OIUIOUA 58

13. A method according to claim 9, wherein the Soxl control sequence comprises nucleotides 1 to 60 of SEQ ID No. 3.

14. A method according to claim 10, wherein the element transactivatable by SOX1 comprises the sequence motif A/T A/T CAA A/T G. A method for producing a cell committed to the neuronal lineage, comprising the steps of: transfecting a pluripotent stem cell with a genetic construct comprising a coding sequence expressing Soxl; culturing the stem cells in order to differentiate them into neural cells; and isolating the neural cells thereby produced.

16. A method according to claim 15, wherein the Soxl sequence is operatively linked to an inducible promoter.

17. A method according to claim 15 or claim 16, wherein the cell is further transfected with a vector comprising a sequence encoding a regulator which modulates the expression of the Soxl sequence.