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  • C12Q2600/158—Expression markers

Definitions

• the present invention relates to a method of marking, selecting and generating neuronal stem sells from tissues.
• 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 their 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 bend within the DNA helix. SOX proteins can induce transcription of reporter constructs in vitro and display properties of both classical transcription factors and architectural components of chromatin (reviewed by Peveny and Lovell-Badge (1997) Curr. Opin. Genetics and Development, 7:338-344).
• Sox gene family are expressed in a variety of embryonic and 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 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. Xs ⁇ x 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 profiles during vertebrate embryogenesis that suggest the genes could function in the control of cell fate decisions within the early developing nervous system. Sox2 and Sox3 begin to be expressed at pre implantation 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.
• a number of transcription factors have been isolated which play important roles in the specification and differentiation of neural cell lineages.
• 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.
• the expression of basic helix-loop-helix transcription factors of the AS- C complex confirms neural potential on groups of ectodermal cells. Miss expression of a transcription factors involved in a neural cell fate determination is observed to cause abnormalities in neural development.
• Soxl expression appears only at around the stage of neural induction in the embryo.
• the role of SOX1 in embryogenesis is, however, not known.
• 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.
• a method for isolating a neuroblastic cell from a population of cells comprising the steps of: (a) detecting the expression of the Soxl gene in the cells; and (b) sorting the cells to isolate those cells expressing the Soxl gene.
• Soxl gene which encodes SOXl, 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.
• Soxl is a unique marker for neuroblasts.
• Cells which are identified as expressing this gene 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.
• a suitable label such as a fluorophore conjugated to or part of, for example, an antisense nucleic acid molecule or an immunoglobulin.
• neuroblast cells can be actively sorted from other cell types by detecting the expression of SOXl in vivo using a reporter system.
• the invention provides a method for isolating a neuroblastic cell from a population of cells, comprising the steps of:
• 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.
• 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 SOXl; in other words, no expression of the marker coding sequence should take place in the absence of SOXl.
• the regulatory sequences of the SOXl 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 SOXl binding site.
• the SOXl control elements are used in their entirety; however, other promoter and enhancer elements may be substituted where they remain under the influence of SOXl .
• the selectable marker will only be expressed in neuroblastic cells because only these cells express SOXl, which is required for transcription from the Soxl control sequences.
• the expression system used to express the selectable marker is not leaky and expresses a minimal amount of the marker in the absence of SOXl.
• 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 neuroblastic cells from one or mores pluripotent precursor cell, comprising the steps of:
• 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.
• 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.
• 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.
• 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.
• Soxl control sequences may be used specifically to direct transgene expression in neuroblast cells where this is desired.
• 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.
• 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.
• the present invention relates to a method for isolating, or producing cells which are committed to the neural fate.
• neuroblast refers to any cell or cell line which has commenced differentiation along the neural pathways.
• 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 the treatment of diseases such as neurodegenerative diseases, and neural damage, for example occasioned by trauma.
• 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.
• cell populations derived from tissue cultures may be employed for the isolation of neuroblastic cells.
• SOXl expression is closely associated with the acquisition of neural fate by the ectoderm, both in vitro and in vivo.
• In vitro SOXl 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, Mashl and Wntl.
• NESTIN neuroepithelial markers
• Mashl neuroepithelial markers
• Wntl neuroepithelial markers
• 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.
• SOXl is detected throughout the cells of the neural plate and early neural tube along its entire anteroposterior axis.
• the early and uniform expression of SOXl throughout the presumptive CNS indicates that SOXl 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.
• 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.
• nucleotide sequences which are substantially the same share at least about 90% identity.
• splice variants having e.g. an additional exon sequence homology may be lower.
• nucleic acids of the invention are preferably substantially homologous to the sequence of human Soxl as shown in SEQ ID No. 3.
• homology means that the two entities share sufficient characteristics for the skilled person to determine that they are similar in origin and function.
• homology is used to refer to sequence identity.
• 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 may be determined using any suitable homology algorithm, using for example default parameters.
• the BLAST algorithm is employed, with parameters set to default values.
• the BLAST algorithm is described in detail at http://www.ncbi.nih.gov/BLAST/blast_help.html, which is incorporated herein by reference.
• the search parameters are defined as follows, and are advantageously set to the defined default parameters.
• substantially homology when assessed by BLAST equates to sequences which match with an EXPECT value of at least 7, preferably at least 9 and most preferably 10 or more.
• the default threshold for EXPECT in BLAST searching is ususally 10.
• BLAST Basic Local Alignment Search Tool
• blastp, blastn, blastx, tblastn, and tblastx these programs ascribe significance to their findings using the statistical methods of Karlin and Altschul (see http://www.ncbi.nih.gov/BLAST/blast_help.html) with a few enhancements.
• the BLAST programs were tailored for sequence similarity searching, for example to identify homologues to a query sequence. 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.
• 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; tblastn compares a protein query sequence against a nucleotide sequence database dynamically translated in all six reading frames (both strands).
• tblastx compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
• BLAST uses the following search parameters:
• HISTOGRAM Display a histogram of scores for each search; default is yes. (See parameter H in the BLAST Manual).
• DESCRIPTIONS Restricts the number of short descriptions of matching sequences reported to the number specified; default limit is 100 descriptions. (See parameter V in the manual page). 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). 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 (e.g., hits against common acidic-, basic- or proline-rich regions), leaving the more biologically interesting regions of the query sequence available for specific matching against database sequences.
• Low complexity sequence found by a filter program is substituted using the letter “N” in nucleotide sequence (e.g., "NNNNNNNNNNNNNNN”) and the letter “X” in protein sequences (e.g. , "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.
• NCBI-gi Causes NCBI gi identifiers to be shown in the output, in addition to the accession and/or locus name.
• sequence comparisons are conducted using the simple BLAST search algorithm provided at http://www.ncbi.nlm.nih.gov/BLAST.
• the invention makes use of fragments of the Soxl -encoding sequence. Fragments of the nucleic acid sequence of a few nucleotides in length, preferably 5 to 150 nucleotides in length, are especially useful as probes.
• 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. 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 1.5°C 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.
• high stringency refers to conditions that permit hybridisation of only those nucleic acid sequences that form stable hybrids in 1 M Na-f- at 65-68 °C.
• 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.
• high stringency washing may be done in several steps, with a final wash (about 30 min) at the hybridisation temperature in 0.2 - O.lx SSC, 0.1 % SDS.
• Low stringency refers to conditions equivalent to hybridisation in the above described solution at about 50-52°C. In that case, the final wash is performed at the hybridisation temperature in 2x SSC, 0.1 % SDS.
• the invention moreover provides nucleic acid sequence which are capable of hybridising, under stringent conditions, to a fragment of SEQ. ID. No. 3.
• the fragment is between 15 and 50 bases in length.
• it is about 25 bases in length.
• 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 SOXl as described below.
• An advantage of the use of a sequence encoding human SOXl 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 SOXl are set forth in SEQ. ID. No. 5.
• nucleic acids encoding Soxl are obtainable according to methods well known in the art.
• 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.
• PCR polymerase chain reaction
• Chemical methods for synthesis of a nucleic acid of interest 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 potential nucleic acid sequences using known and preferred coding residues for each amino acid residue.
• cDNA expression libraries are screened with probes or analytical tools designed to identify the gene of interest or the protein encoded by it.
• 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 screemng 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.
• 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 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.
• nucleic acid probes of the invention are labelled with suitable label means for ready detection upon hybridisation.
• suitable label means is a radiolabel.
• the preferred method of labelling a DNA fragment is by incorporating ⁇ 32 P 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 ⁇ 32 P-labelled ATP and polynucleotide kinase.
• other methods e.g. non-radioactive
• 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 (i.e., 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.
• 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. 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 SOXl mutant that has an amino acid sequence differing from the SOXl 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 may be performed by any technique known in the art, as exemplified above.
• the cells may be sorted by flow cytometry or FACS.
• flow cytometry FACS
• FACS Fluorescence Activated Cell Sorting
• F.A.C.S. Fluorescence Activated Cell Sorting
• flow cytometry Fluorescence Activated Cell Sorting
• 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 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.
• fluorescent dyes such as fluorescein and phycoerythrin.
• direct immunofluorescence an antibody to a particular molecule, the SOXl polypeptide, is directly conjugated to a fluorescent dye. Cells can then be stained in one step.
• 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-SOXl 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 SOXl . This can be achieved directly, by labelling of the protein product, or indirectly by using a reporter gene in the construct.
• reporter genes are ⁇ -galactosidase and Green Fluorescent Protein (GFP).
• ⁇ - galactosidase activity can be detected by FACS using fluorogenic substrates such as fluorescein digalactoside (FDG).
• FDG fluorescein digalactoside
• FDG fluorescein digalactoside
• FDG fluorescein digalactoside
• 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.
• 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 teaching set forth above, using the general procedures as described by Sambrook et al (1989).
• 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, liposome- mediated transfer of nucleic acids or imaging agents, and the like.
• the invention in certain embodiments, includes antibodies specifically recognising and binding to SOXl.
• such antibodies may be generated against the SOXl having the amino acid sequences set forth in SEQ ID No. 4.
• SOXl or SOXl 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 SOXl epitope.
• Anti-SOXl 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 SOXl 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 therapeutic applications on account of their small size and consequent superior tissue distribution.
• the antibodies may comprise a label.
• 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.
• they may be fluorescent labels or other labels which are visualisable in tissues and which may be used for cell sorting.
• chimeric antibodies may be constructed in order to decrease the immunogenicity thereof in diagnostic or therapeutic applications.
• 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.
• 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.
• a host e.g. E. coli or a mammalian cell
• 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 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.
• suitable culture media which are the customary standard culture media, for example Dulbecco's Modified Eagle Medium (DMEM) or RPMI 1640 medium
• a mammalian serum e.g. foetal calf serum
• trace elements and growth sustaining supplements e.g. feeder cells
• feeder cells such as normal mouse peritoneal exudate cells, sple
• 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 scale- up 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.
• the desired antibodies can also be obtained by multiplying mammalian cells in vivo.
• hybridoma cells producing the desired antibodies are injected into histocompatible mammals to cause growth of antibody- producing tumours.
• the animals are primed with a hydrocarbon, especially mineral oils such as pristane (tetramethyl-pentadecane), prior to the- injection.
• pristane tetramethyl-pentadecane
• 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.
• the cell culture supernatants are screened for the desired antibodies, preferentially by immunofluorescent staining of cells expressing SOXl, by immunoblotting, by an enzyme immunoassay, e.g. a sandwich assay or a dot-assay, or a radio immunoassay .
• 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.
• 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 SOXl 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 SOXl, characterised in that a suitable mammal, for example a Balb/c mouse, is immunised with purified SOXl protein, an antigenic carrier containing purified SOXl or with cells bearing SOXl, antibody- producing 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.
• a suitable mammal for example a Balb/c mouse
• spleen cells of Balb/c mice immunised with cells bearing SOXl 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.
• 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 SOXl containing a suitable adjuvant several times, e.g. four to six times, over several months, e.g.
• 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.
• a fusion promoter preferably polyethylene glycol.
• 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.
• 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 SOXl as described hereinbefore.
• 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.
• DNA encoding a heavy chain variable domain and/or for a light chain variable domain of antibodies directed SOXl 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.
• 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 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 light chain murine variable domain.
• mutant is intended to include a DNA mutant obtained by in vitro mutagenesis of the authentic DNA according to methods known in the art.
• 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 SOXl fused to a human constant domain g, for example ⁇ l, ⁇ 2, ⁇ 3 or ⁇ 4, preferably ⁇ l or ⁇ 4.
• the invention concerns recombinant DNAs comprising an insert coding for a light chain murine variable domain of an antibody directed to SOXl fused to a human constant domain K or ⁇ , preferably K.
• 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 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.
• 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 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.
• neuroblastic cells may be actively sorted from other cell types by detecting Soxl expression in vivo using a reporter system.
• 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.
• 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.
• the Soxl control sequences used are human Soxl control sequences. Preferably, they comprise nucleotides 1 to 60 of SEQ. ID. No. 3.
• reporter constructs useful for detecting neural cells by expression of a reporter gene may be constructed the general teaching of Sambrook et al (1989).
• 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.
• 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.
• constructs according to the invention 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.
• the present invention relates to the transfection of pluripotent precursor cells, capable of differentiating into neural cells, with a vector expressing Soxl.
• pluripotent precursor cells may be induced to differentiate along the neural pathway, becoming precursor neurons capable of differentiating into a variety of neural tissues.
• transfection transfection
• transformation transformation
• 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.
• ES cells such as human ES cells and cells derived from a Germ cells (EG cells) may be derived from embryonal tissue.
• 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., (1996) Clinical Neuroscience 3:301-309.
• 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.
• vector refers to discrete elements that are used to introduce heterologous DNA into cells for expression. Selection and use of such vehicles are 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.
• 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.
• an expression and cloning vector may contain a selection gene, also referred to as selectable marker, other than that intended for marking Soxl- expressing 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. 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 SOXl, 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 SOXl by removing the promoter from the source DNA and inserting the isolated promoter sequence into the vector. Both the native SOXl promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of SOXl 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.
• any suitable promoter may be used, when placed under the control of a SOXl -inducible element.
• 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 SOXl or the label.
• An expression vector includes any vector capable of expressing SOXl or label- encoding nucleic acids that are operatively linked with regulatory sequences, such as promoter regions, that are capable of expression of such DNAs.
• 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.
• DNAs encoding SOXl 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).
• 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.
• transient expression systems are useful e.g. for identifying SOXl expressing cells or for inducing a pluripotent cell to differentiate.
• Plasmids 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.
• MATERIAL AND METHODS Manufacture of SOXl polyclonal antibodies A 622bp Hindi fragment encoding sequences C-terminal of the HMG box of SOXl (207 a. a.) 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 67:31-40. rabbits are treated with a course of injections as recommended by Smith and Johnson (1988): each injection contains 250 ⁇ g 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-SOXl PAb (1:500); K2 anti- HNF3 ⁇ MAb (1 :40); 6G3 anti-FP3 MAb (1: 10); anti-3A10 MAb (1 : 10); anti- 2H3(Neurofilament-160) MAb (1 : 10); 4D5 anti-Islet-1 MAb (1 : 1000); anti-SSEAl 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.
• FITC fluorescein isothio
• BrDU analysis Pregnant mice are injected intraperitoneally with 50 ⁇ g/g of body weight of 5-bromo-2deoxyuridine (BrDU) (Sigma) in 09. % NaCl and sacrificed two hours after injection. Embryos are fixed and sectioned as described above. The slides are washed twice in PBS, and incubated in 0.2% HCl at 37°C for 30 minutes, then rinsed thoroughly with PBS, followed by three rinses with PBS/0.1 % Trinton/1 % heat inactivated goat serum (P-T-G). Monoclonal anti-BrDU (1:500 dilution in P-T-G) is applied to the sections and incubated at 4°C overnight.
• PrDU 5-bromo-2deoxyuridine
• Sequential sections are incubated in SOXl 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 l ⁇ M retinoic acid or in the presence of l ⁇ M retinoic acid or in the presence of 5mM 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 lx MEMFA for 1 hour, washed in P-T-G twice; then stained with appropriate antibody.
• Plasmids and transfection To construct the SOXl expression vector, pRSVopS ⁇ xi, 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 ⁇ g/ml of hygromycin. Expanded clones (250) are isolated and examined for expression of the Lac repressor by indirect immunofluorescence with anti-lac PAb (Stratagene).
• P3'SS-10 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 pRSVopS ⁇ xi by lipofection. Stable clones are selected using 500 ⁇ g/ml G481. 250 clones are expanded and analysed for inducible Soxl expression by RNase protection and immunocytochemistry with SOXl antibody.
• RNA is prepared from P19 cells and RNase protection assays are carried out using 5 ⁇ g 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 Smal-BspHl 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 Notl digest of SAP D cDNA is used a loading control (Dresser et al., (1995) Hum. Mol. Genet. 4:1613-1618).
• RNA 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 froih 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) EXAMPLE 1
• SOXl expression during mouse and rat neurulation is analysed using a rabbit polyclonal antibody against the SOXl C-terminal region.
• expression of SOXl 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.
• SOXl expression at this stage is specific to the neural plate.
• SOXl is maintained in all neuroepitheial cells along the entire anteroposterior axis as the neural pate bends (8.0-8.5 dpc.
• D/V dorsoventral
• Soxl is downregulated in a stereotyped manner in cells alone D/V axis of the neural tube.
• the SOXl antibody is used in combination with a panel of antigenic markers which identify cells of the floor plate and mature neurons (Neurofilament (NF-1): labelled with contrasting colour markers and visualised in an El l rat embryo). Expression of SOXl and expression of these markers is almost entirely mutually exclusive. In the ventral spinal cord or the 10.0-12.0 dpc mouse embryo, SOXl 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.
• SOXl expression is restricted to a thin ventricular zone in the CNS.
• SOXl expression in to detected in the peripheral nervous system (PNS).
• PNS peripheral nervous system
• 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 mitosis, migrate to the outer layer, the marginal zone (MZ).
• VZ inner ventricular zone
• MZ marginal zone
• SOXl expression is detected, using an anti-SOXl 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.
• BrDU bromodeoxyuridine
• 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 SOXl expression. Similar to SOXl 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 SOXl. SOXl-positive cells that do not incorporate BrDU are restricted to the luminar surface of the ventricular zone. In contrast, no SOXl nor BrDU-positive cells are detected in the outer marginal zone. These results show that SOXl is expressed in dividing neuroepithelial cells within the embryonic CNS.
• HNF3 ⁇ thus provides a marker for cells that are mitotically active but have begun to differentiate.
• 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 SOXl in neural cells together with observations that Sox genes may affect cell lineage decisions, raises the possibility that SOXl expression is an early response to neural inducing signals and that its expression may be involved in directing cells towards a neural fate.
• SOXl plays a role in establishing neural fate in response to A P19 cell culture system is used as an in vivo model system in which to analyse SOXl 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-
• Soxl 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-SOXl and anti-SSEA antibodies, and by RNase protection.
• SOXl when P19 cells are differentiated as aggregates without the addition of chemical inducers, SOXl is not expressed as determined by RNase protection.
• SOXl 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.
• retinoic acid-treated PI 9 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 SOXl and Neurofilament, to examine the expression of SOXl in P19 cells displaying a fully differentiated neuronal morphology.
• SOXl immunoreactivity is not detected in process-bearing Neurofilament-positive neurons.
• SOXl is expressed by P19 cells when they first assume a neural fate but it is then downregulated with their differentiation.
• This parent line (P3'SS-10) is transfected with pRSVopSoxi, a vector containing the Soxl cDNA under the regulation of an inducible RSV promoter and stable lines are established.
• IPTG isopropyl- ⁇ -d- thiogalactase
• 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 .
• IPTG isopropyl- ⁇ -d- thiogalactase
• 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 RSVopSoxi 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).
• 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 SOXl both in vivo and in vitro, is mutually exclusive with mature neuronal markers such as Neurofilament and Isletl.
• double-label immunoflourescence is used to simultaneously detect SOXl and Neurofilament. No SOXl expression could be detected in cells positive for Neurofilament in these cultures.
• MOLECULE TYPE cDNA to mRNA
• HYPOTHETICAL NO
• ANTI-SENSE NO
• CAAGATGCAC AACTCGGAGA TCAGCAAGCG GCTGGGCGCC GAGTGGAAGG TGATGTCGGA 480 GGCCGAGAAG CGGCCTTTCA TCGACGAGGC GAAGCGGCTG CGGGCGCTGC ACATGAAGGA 540
• CTCGCAGCCC CACCAGAACT CGGCGGCCGC GGCGGCGGCG GCGGCGGCGG CGGCGGCCGC 1080 CTCGTCGGGC GCGCTGGGCG CGCTGGGCTC GCTCGTCAAG TCGGAGCCCA GCGTGAGCCC 1140
• CTCCCTGCCC CAGCACTACC AGAGCGCCAG CACGGGGGTC AACGGCACCG TCCCCTTGAC 1320
• GCACATCTGA GCGGCCCCGG AGCGGCCCCG GAGCGGCGCG GAGGGCCCCG GCCCGGGCCC 1380 CGCAGGACTG CGGCCCCGCC GCCGCCCCGC GCCCGCCGCC CCCCTTCGTT TTTGCCTTTC 1440
• ATTCGGCTCC TTCCCGCCCT CCCCCTCCCT CCTTCCTTTT TTTGTTTTGT TTTGTTTTGT 1500
• TTTTCTTTTC TTCCTTTTTG TACAGAAATG TTTTGATGTT CTTGTAATAA TAATAAATAA 1560
• TTCCTCGCAC ACCCCAAAAC AGCACCACGA GTTTCCGTAG ATGTTCTCGC GCTTTTCCTT 1980 TTTGGTTGGG TTATTTCGGC TGCTTTATTT ATACAACTTT TTCTTCTTCT TCCTTTCTTC 2040 CCGAGGTTGC AACGTTTGCT TGATTTTTAT TTTATTTTAT TTTTTTTTCT GGGTTATG 2100
• TTTTTTTACA TTTTTTTGTA TCATCTCGTG TAAATGCATT GTGAAATAAT TTTTATCTAG 2220
• MOLECULE TYPE cDNA to mRNA
• HYPOTHETICAL NO
• ANTI- SENSE NO
• ORIGINAL SOURCE
• ORGANISM Mus musculus
• CAGGGCGCGG GCGCGGGCGT CAACGGCACG GTGCCCCTGA CGCACATCTA GCGCCGCGGG 1620 GACGCCGGGG ACACTGCGGC TTAAGGCCGG CGCCCCGGCG ACGAAGAGCG AGGCCTGCGC 1680
• MOLECULE TYPE cDNA to mRNA
• HYPOTHETICAL NO
• ATC AGC AAG CGC CTG GGG GCC GAG TGG AAG GTC ATG TCC GAG GCC GAG 347 lie Ser Lys Arg Leu Gly Ala Glu Trp Lys Val Met Ser Glu Ala Glu 85 90 95 AAG CGG CCG TTC ATC GAC GAG GCC AAG CGG CTG CGC GCG CTG CAC ATG 395 Lys Arg Pro Phe lie Asp Glu Ala Lys Arg Leu Arg Ala Leu His Met 100 105 110
• Lys Arg Pro Phe lie Asp Glu Ala Lys Arg Leu Arg Ala Leu His Met 100 105 110
• Ser Pro lie Ser Asn Ser Gin Gly Tyr Met Ser Ala Ser Pro Ser Gly 260 265 270 Tyr Gly Gly Leu Pro Tyr Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala His 275 280 285
• Arg Glu Met lie Ser Met Tyr Leu Pro Ala Gly Glu Gly Gly Asp Pro 340 345 350 Ala Ala Ala Ala Ala Ala Ala Ala Ala Gin Ser Arg Leu His Ser Leu Pro 355 360 365
• UURUURGCNG CNGGNGCNGG NGGNGGNGGN
• GCNGCNGUNG CNAUGGGNGU NGGNGUNGGN 480
• GGNGCNCAYC CNCAYAGRAC NCCNGCNCAY CCNCAYCCNC AYCAYCCNCA YGCNCAYCCN 720
• MOLECULE TYPE DNA (genomic)

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• 1997
  • 1997-06-25 GB GBGB9713469.6A patent/GB9713469D0/en active Pending
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