Use of regulatory sequences for specific, transient expression in neuronal determined cells

Abstract

The present invention relates to the use of regulatory sequences for mediating specific, early transient expression in proliverative neuronal determined cells. Furthermore, the uses of recombinant nucleic acid molecules comprising said defined regulatory sequences for mediating specific, early transient expression in proliverative neuronal determined cells as well as for the generation of non-human transgenic organisms and/or host cells are disclosed. In addition, the invention provides for transgenic non-human animals and/or host cells comprising said regulatory sequences and/or recombinant nucleic acid molecules. The invention also describes methods for the preparation of such vectors, host cells and transgenic nonhuman animals as well as methods for the detection and/or isolation of neuronal determined cells. Additionally, methods for screening of compounds capable of regulating neuronal determined cell activity, neurogenesis, stimulating proliferation of neuronally committed precursor cells and/or neuronal differentiation are provided and the invention also relates to methods for the detection and analysis of neuronal differentiation, neuronal migration and/or neuronal determination processes. Finally, the invention relates to diagnostic and pharmaceutical compositions comprising the regulatory sequences, recombinant nucleic acid molecules, host-cells or isolated neuronal determined cells described herein.

1 - 56 . (canceled)  
     
     
         57 . A DNA segment comprising a regulatory sequence isolated free of the complete DCX gene protein coding region, wherein the regulatory sequence comprises a regulatory sequence selected from the group consisting of: 
 (a) regulatory sequences comprising the nucleotide sequence shown in SEQ ID NO: 1, as shown in SEQ ID NO: 2, as shown in SEQ ID NO: 3 or as shown in SEQ ID NO: 4;    (b) regulatory sequences comprising the nucleotide sequence contained in the insertion of clone DSM 15111 and obtainable by amplification using two oligonucleotides having the sequences indicated under SEQ ID NO: 9 and SEQ ID NO: 10;    (c) regulatory sequences comprising at least one nucleotide sequence of SEQ ID NO: 1 from position 1166 to 1746, from position 1166 to 2049, from position 1785 to 1843 or from position 1953 to 2775;    (d) regulatory sequences comprising at least one nucleotide sequence of SEQ ID NO: 2 from position 529 to 1079, from position 529 to 1390, from position 1118 to 1175 or from position 1291 to 2137;    (e) regulatory sequences comprising at least a functional part of a sequence of (a) to (d) and causing specific expression in neuronal determined cells;    (f) regulatory sequences comprising a nucleotide sequence which is at least 75% identical to a sequence as defined in (a) to (d) or which comprises a nucleotide sequence which is at least 78% identical to the nucleotide sequence as shown in SEQ ID NO: 1 from position 1166 to 1746 or from position 1166 to 2049 or to the nucleotide sequence shown in SEQ ID NO: 2 from position 529 to 1079 or from position 529 to 1390, which comprises a nucleotide sequence which is at least 82% identical to the nucleotide sequence as shown in SEQ ID NO: 1 from position 1785 to 1843 or to the nucleotide sequence as shown in SEQ ID NO: 2 from position 1118 to 1175 or which comprises a nucleotide sequence which is at least 75% identical to the nucleotide sequence as shown in SEQ ID NO: 1 from position 1953 to 2775 or to the nucleotide sequence as shown in SEQ ID NO: 2 from position 1291 to 2137; and    (g) regulating sequences comprising a nucleotide sequence which hybridizes with a complementary strand of the regulatory sequence as defined in (a) to (f) for the early, transient expression of a heterologous nucleotide sequence in proliferative neuronal determined cells.    
     
     
         58 . The DNA segment of  claim 57 , wherein said DNA segment further comprises a heterologous nucleotide sequence to be expressed.  
     
     
         59 . The DNA segment of  claim 57 , wherein said regulatory sequence is of human, mouse or rat origin.  
     
     
         60 . The DNA segment of  claim 58 , wherein said heterologous nucleotide sequence to be expressed is a gene selected from the group consisting of a marker gene, a receptor gene, an anti-apoptotic gene, a determination/differentiation gene, a gene capable of inducing and/or directing neuronal migration or guidance, a gene encoding a tag, a gene encoding for a trophic factor, a gene encoding a surface protein, a gene encoding for a transcription factor or a gene encoding an enzyme, or wherein said nucleotide sequence to be expressed is an antisense sequence or encodes for a ribozyme or an inhibiting RNA molecule.  
     
     
         61 . The DNA segment of  claim 60 , wherein said marker or receptor gene is selected from the group consisting of GFP, EGFP, RFP, CFP, BFP, YFP, DsRed, f3-galactosidase, luciferase or chloramphenicol acetyltransferase.  
     
     
         62 . The DNA segment of  claim 60 , wherein said gene encoding a tag is selected from the group consisting of a His-Tag, glutathione, a Strep-tag, a Flag-tag, CBP, TAG-100, E2-tag and Z-tag.  
     
     
         63 . The DNA segment of  claim 60 , wherein said gene encoding a surface protein is CD24.  
     
     
         64 . The DNA segment of  claim 60 , wherein said gene encoding for a trophic factor is selected from the group consisting of NGF, BDNF, PDGF, NT-3, NT-4, NT-5, VEGF, PEDF, EGF, FGF, IGF, cardiotrophin, erythropoietin, leptin, LIF, and TGF.  
     
     
         65 . The DNA segment of  claim 60 , wherein said anti-apoptotic gene is selected from the group consisting of bcl-2, Brn-3a, PTEN and (an) anti-caspase(s).  
     
     
         66 . The DNA segment of  claim 60 , wherein said determination/differentiation gene is the dopaminergic determination factor Nurr1.  
     
     
         67 . The DNA segment of  claim 60 , wherein said gene capable of inducing and/or directing neuronal migration or guidance is selected from the group consisting of netrin, neuropilin, CXCR4, SDF-1, DCC, slit, robo, a semaphorin, a plexin family member, and an ephrin family member.  
     
     
         68 . The DNA segment of  claim 60 , wherein said gene encoding a transcription factor is selected from the group consisting of NeuroD, BMP4, Nurr1 or ShcC.  
     
     
         69 . The DNA segment of  claim 60 , wherein said gene encoding an enzyme is CRE.  
     
     
         70 . The DNA segment of  claim 60 , wherein said heterologous nucleotide sequence to be expressed is an antisense sequence, or encodes for a ribozyme or an inhibiting RNA-molecule.  
     
     
         71 . The DNA segment of  claim 70 , wherein said inhibiting RNA is selected from the group consisting of RNAi, siRNA, shRNA and stRNA.  
     
     
         72 . The DNA segment of  claim 58 , wherein said DNA segment is further defined as a recombinant vector comprising said a regulatory sequence and heterologous nucleotide sequences to be expressed positioned under the control of said regulatory sequence.  
     
     
         73 . The DNA segment of  claim 72 , wherein said vector is further defined as a virus.  
     
     
         74 . The DNA segment of  claim 72 , wherein said vector is further defined as vector is suitable for gene therapy or vaccination with a nucleic acid molecule.  
     
     
         75 . The DNA segment of  claim 57 , wherein the regulatory sequence comprises the nucleotide sequence shown in SEQ ID NO: 1, as shown in SEQ ID NO: 2, as shown in SEQ ID NO: 3 or as shown in SEQ ID NO: 4.  
     
     
         76 . The DNA segment of  claim 57 , wherein the regulatory sequence comprises the nucleotide sequence contained in the insertion of clone DSM 15111 and obtainable by amplification using two oligonucleotides having the sequences indicated under SEQ ID NO: 9 and SEQ ID NO: 10.  
     
     
         77 . The DNA segment of  claim 57 , wherein the regulatory sequence comprises at least one nucleotide sequence of SEQ ID NO: 1 from position 1166 to 1746, from position 1166 to 2049, from position 1785 to 1843 or from position 1953 to 2775.  
     
     
         78 . The DNA segment of  claim 57 , wherein the regulatory sequence comprises at least one nucleotide sequence of SEQ ID NO: 2 from position 529 to 1079, from position 529 to 1390, from position 1118 to 1175 or from position 1291 to 2137.  
     
     
         79 . The DNA segment of  claim 57 , wherein the regulatory sequence comprises a nucleotide sequence which is at least 75% identical to the regulatory sequences set forth in SEQ ID NOs 1 through 4, or which comprises a nucleotide sequence which is at least 78% identical to the nucleotide sequence as shown in SEQ ID NO: 1 from position 1166 to 1746 or from position 1166 to 2049 or to the nucleotide sequence shown in SEQ ID NO: 2 from position 529 to 1079 or from position 529 to 1390, which comprises a nucleotide sequence which is at least 82% identical to the nucleotide sequence as shown in SEQ ID NO: 1 from position 1785 to 1843 or to the nucleotide sequence as shown in SEQ ID NO: 2 from position 1118 to 1175 or which comprises a nucleotide sequence which is at least 75% identical to the nucleotide sequence as shown in SEQ ID NO: 1 from position 1953 to 2775 or to the nucleotide sequence as shown in SEQ ID NO: 2 from position 1291 to 2137.  
     
     
         80 . The DNA segment of  claim 57 , wherein the regulatory sequence comprises a nucleotide sequence which hybridizes with a complementary strand of the regulatory sequence as defined in  claim 78  under stringent conditions and which provided for the early, transient expression of a heterologous nucleotide sequence in proliferative neuronal determined cells.  
     
     
         81 . A host cell comprising a DNA segment in accordance with  claim 58 .  
     
     
         82 . The host cell of  claim 81 , wherein said DNA segment is positioned such that the heterologous nucleotide sequence is expressible by said cell.  
     
     
         83 . The host cell of  claim 81 , whereis said cell is further defined as neuronal cell, an ES-cell, a germ cell, a cultured cell or a primary cell.  
     
     
         84 . The host cell of  claim 80 , further defined as a neuronal determined cell.  
     
     
         85 . A method of expressing a heterologous nucleotide sequence in a host cell comprising obtaining a host cell in accordance with  claim 82  and culturing said host cell in a manner effective to achieve the expression of said heterologous nucleotide sequence.  
     
     
         86 . The method of  claim 85 , wherein the host cell is a neuronal determined cell and the method is further defined as a method for the early expression of the heterologous nucleotide sequence in the neuronal determined cell.  
     
     
         87 . The method of  claim 85 , wherein said host cell comprises a neuronal cell, an ES-cell, a germ cell, a cultured cell or a primary cell.  
     
     
         88 . The method of  claim 85 , wherein the host cell is further defined as a neuronal determined cell.  
     
     
         89 . A method of expressing a nucleotide sequence to be expressed comprising positioning such a nucleotide sequence under the control of a regulatory sequence as defined by  claim 57  and expressing such nucleotide sequence in a host cell.  
     
     
         90 . A method of preparing a genetically modified host cell comprising obtaining a regulatory sequence in accordance with  claim 57  and introducing said sequence into a selected host cell to provide a genetically modified host cell.  
     
     
         91 . A method for the generation of a non-human transgenic animal comprising the step of introducing a regulatory sequence as defined in claim  1  into an ES-cell or a germ cell.  
     
     
         92 . A non-human transgenic animal which comprises host cells as defined by  claim 81 .  
     
     
         93 . The non-human transgenic animal of  claim 92 , wherein said transgenic animal is a mouse or a rat.  
     
     
         94 . The non-human transgenic animal of  claim 92 , which comprises a nucleotide sequence under the control of said regulatory sequence which encodes for GFP, EGFP, RFP, BFP, YFP, 13-galactosidase, chloramphenicol, acetyltransferase or luciferase.  
     
     
         95 . A composition comprising the regulatory sequence as defined in claim  1 , a recombinant expression molecule comprising a nucleotide sequence to be expressed positioned under the control of such regulatory sequence, or a vector or host cell comprising such a recombinant expression molecule.  
     
     
         96 . The composition of  claim 95 , which is a pharmaceutical composition, optionally comprising a pharmaceutically acceptable carrier or diluent.  
     
     
         97 . The composition of  claim 95 , which is a diagnostic composition, optionally further comprising suitable means of detection.  
     
     
         98 . A method for the detection of (a) proliferative neuronal determined cell(s) comprising the steps of: 
 (a) expressing in a plurality of cells a recombinant nucleic acid molecule as defined in  claim 58;  and    (b) selecting cells which express a heterologous nucleotide sequence under the control of the regulatory sequence.    
     
     
         99 . The method of  claim 98 , which comprises in step (b) the detection of an expressed, heterologous marker or reporter gene.  
     
     
         100 . The method of claim  42  which comprises the detection of a fluorescent marker or reporter or an expressed enzyme.  
     
     
         101 . The method of  claim 99 , which comprises a FACS analysis, MACS analysis or affinity isolation.  
     
     
         102 . The method of  claim 99 , further comprising separating the cell(s) which express(es) a heterologous nucleotide sequence under the control of the regulatory sequence from (a) cell(s) which do not express said heterologous nucleotide sequence or which comprises an additional step of separating (a) cell(s) which comprise an activated regulatory sequence.  
     
     
         103 . A method for screening of compounds capable of regulating neural stem cell activity, neurogenesis and/or neuronal differentiation comprising the steps of: 
 (a) obtaining a compound that is to be tested for its ability to regulate neural stem cell activity, neurogenesis and/or neuronal differentiation;    (b) contacting a DNA segment in accordance with  claim 58 , or a cell comprising such a DNA segment, with said compound; and    (c) detecting whether said compound(s) is/are capable of interacting with said regulatory sequence.    
     
     
         104 . A method for the preparation of a pharmaceutical composition for the treatment of a neurological disorder or disease comprising the steps of the method of  claim 103  whereby said compound capable of regulating neural stem cell activity, neurogenesis and/or neuronal differentiation obtained by said method is further formulated with a pharmaceutically acceptable carrier, excipients and/or diluent.