EP0977778A2 - Promotoren von exon 1 und exon 3 des menschlichen nervenwachstumsfaktors. - Google Patents

Promotoren von exon 1 und exon 3 des menschlichen nervenwachstumsfaktors.

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Publication number
EP0977778A2
EP0977778A2 EP98901211A EP98901211A EP0977778A2 EP 0977778 A2 EP0977778 A2 EP 0977778A2 EP 98901211 A EP98901211 A EP 98901211A EP 98901211 A EP98901211 A EP 98901211A EP 0977778 A2 EP0977778 A2 EP 0977778A2
Authority
EP
European Patent Office
Prior art keywords
promoter
exon
growth factor
nerve growth
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98901211A
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English (en)
French (fr)
Inventor
Matthew D. Linnik
Margaret M. Racke
Joan M. Krakowsky
Arun Subramaniam
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Aventis Pharmaceuticals Inc
Original Assignee
Hoechst Marion Roussel Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hoechst Marion Roussel Inc filed Critical Hoechst Marion Roussel Inc
Publication of EP0977778A2 publication Critical patent/EP0977778A2/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/48—Nerve growth factor [NGF]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

  • NGF nerve growth factor
  • NGF can also help to sustain function in aged or damaged neurons by maintaining neuronal phenotype and inducing neurite outgrowth (Fischer W, et al., Nature 329:65-68, 1987; Fischer W, et al, J Neurosci 11 :1889-1906, 1991; Rylett, R.J., et al., J Neurosci 13:3956-3963, 1993; Chen KS, et al., Neuroscience 68(1): 19- 27, 1995; Tuszynski MH and Gage FH, Mol Neurobiol 10:151-167, 1995).
  • NGF nerve growth factor
  • Several alternative routes of administration have proven effective, including direct intracerebroventricular administration, implantation of producer cell lines (Rosenberg MB, et al., Science 242:1575-1578, 1988), conjugation to actively transported molecules (Friden PM, et al., Science 259:373-377, 1993; Kordower JH, et al., PNAS USA Sep 13; 91(19): 9077-80, 1994) and transcriptional upregulation by low molecular weight compounds.
  • NGF-inducing compounds have been shown to upregulate NGF mRNA transcription via the two promoter regions which have been identified in the mouse NGF gene (Selby MJ, et al., Molec Cell Biol 7:3057-3064, 1987; Nitta A., et al., Eur J Pharmacol 250:23-30, 1993). Recently, a third promoter has been suggested in the rat NGF gene (Timmusk T, et al., Soc Neurosci Absts 21 :33, 1995).
  • the mouse promoter at exon 1 has been well studied and a functional AP-1 regulatory element has been described 35 bases 3' of the start of exon 1 (D'Mello SR, and Heinrich G. J Neurochem 57:1570-1576, 1991; D'Mello SR, and Heinrich G., Molec Cell Neurosci 2:157- 167, 1991; Cowie A, et al., Mol Brain Res 27:58-62, 1994).
  • An identical element exists in the human gene at the same location (Cartwright M, et al., Mol Brain Res 15:67-75, 1992).
  • the regulation of the human and mouse NGF promoters is not identical.
  • functional analyses of the human gene revealed a 5' consensus AP-1 site at -74 in the human gene that is not present in the mouse gene (Cartwright M, et al., Mol Brain Res 15:67-75, 1992).
  • the 3' intron 1 AP-1 site is present in humans and rodents and is also thought to be involved in basal expression, lesion induced increases in NGF mRNA and phorbol ester responsiveness (D'Mello SR, and Heinrich G., Molec Cell Neurosci 2:157-167, 1991; Cowie A, et al., Mol Brain Res 27:58-62, 1994; Hengerer B, et al., Proc. Natl. Acad. Sci. USA 87:3899-3903 (1990).
  • phorbol 12-myristate 13 -acetate enhances the synthesis of NGF in mouse L929 fibroblasts and in primary glial cells (D'Mello SR, and Heinrich G. J Neurochem 55:718-721, 1990; Wion D, et al., FEBS Lett 262:42-44, 1990; Neveu I, et al., Brain Res 570:316-322, 1992) but suppresses expression in ROS 17/2.8 osteoblastic cells (Jehan F, et al., Molec and Cell Endocrinol 116:149-156, 1996).
  • astrocytes as a source of NGF in vivo, particularly after a traumatic insult.
  • Lee TH et al., Brain Res 713:199-210, 1996; Kossmann T, et al., Brain Res 713:143-152, 1996; DeKosky ST, et al., Ann Neurol 39:123-7, 1996) and it has been recognized that glial derived cell lines can synthesize and secrete nerve growth factor (Carman-Krzan M, et al, J-Neurochem 56(2): 636-43, 1991; Lu B, et al.. J-Neurosci 11(2): 318-26, 1991).
  • a number of physiologic changes are known to induce NGF in vivo.
  • a sciatic nerve lesion induces NGF in nonneuronal cells of the sciatic nerve (Lincholm, D. R., et al, Nature 350:658-659 (1987).
  • Transection of fimbria fornix induces NGF in the hippocampus and basal forebrain. (Gasser, U.E., et al., Brain Res. 376:351-356, 1986, Weskamp, G., et al, Neurosci. Lett. 70:121-126, 1986).
  • Electrolytic lesion of the septohippocampal pathway induces NGF in the hippocampus and basal forebrain astrocytes.
  • Seizure activity has been shown to transiently increase mRNA levels of NGF and other neurotrophic factors, such as BDNF, in cortical and hippocampal neurons. These changes are observed after limbic seizures have been induced by a wide variety of insults, such as dentate hilar lesion, kainic acid, or kindling, as well as after injections of bicuculline or pentylenetratrazol. (Lindvall, O., et al., TINS 17(11) 1994:490-496).
  • Alzheimer's disease is a neurodegenerative disease that is partially characterized by progressive loss of cognitive function.
  • Biological changes associated with Alzheimer's disease include formation of amyloid-rich neutic plaques and neurofibrillary tangles in areas associated with learning and memory—the hippocampus and neocortex.
  • Acetylcholine- containing (cholinergic) neurons found in the basal forebrain decrease, and the severity of the cognitive deficit observed in Alzheimer's patients closely correlates with the loss of cholinergic neurons in the basal forebrain.
  • NGF protein and mRNA encoding NGF are localized in the hippocampus and neocortex, the major cholinergic target areas of the basal forebrain neurons. These cholinergic neurons have been shown to shrink and die following damage and with age, possibly due to a loss of target contact with the hippocampus and cortex.
  • NGF neurotrophic factor
  • NGF neurotrophic factor
  • Delivery of exogenous NGF presents some particular challenges. If administered intravenously, NGF is not able to cross the blood-brain barrier and hence is not able to get to the target neurons of the hippocampus or cortex. Admimstration directly into the brain, via a ventricular reservoir or pump, is costly, difficult and exposes the central nervous system to potential infections, as well as being uncomfortable for the patient.
  • a possible solution to delivery problems may be bioactive fragments of NGF, which may have a higher degree of biological activity than NGF and more easily penetrate the blood-brain barrier. Smaller fragments may also be more cost effective, as they are smaller and easier to prepare recombinantly. However, to date, truncated NGF fragments have not been successfully administered and appear to lose activity.
  • NGF-producing cell lines directly into the site of needed activity.
  • this approach requires genetic manipulation of a cell, which may present significant regulatory approval problems.
  • Many of the host cell lines used e.g., fibroblasts, are possibly tumorgenic and may continue to proliferate after transplantation into the CNS.
  • cell surface markers on the cell line may provoke rejection by the immune system. It is not currently possible to control the level of NGF secretion into the adjacent tissue.
  • Another potential therapeutic approach is upregulation of endogenous NGF production by administration of a small molecule which directly activates transcription of NGF and hence leads to greater NGF mRNA and ultimately increased NGF protein production.
  • small molecules are capable of passing through the blood-brain barrier, and may easily be formulated for either intravenous or oral administration.
  • the present invention is directed to the novel human genomic DNA sequences adjacent to, or within, the NGF gene which contain promoters for NGF transcription.
  • reporter constructs comprising all or part of the DNA sequence provided herein attached to a reporter gene, for example, the luciferase gene, ⁇ -galactosidase or green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • These novel reporter constructs may be then used to screen compounds for their ability to affect transcription of NGF.
  • the present invention is also directed to a method for assaying a compound for its ability to affect transcription of the NGF promoter.
  • Preferred embodiments of nucleic acid of the invention are as follows:
  • An isolated nucleic acid comprising human nerve growth factor exon 1 promoter selected from 1 - 1786, 2274 - 2846, human nerve growth factor exon 3 promoter 1 - 1877, fragment thereof, or modified form thereof.
  • nucleic acid according to 1 wherein the nucleic acid is nerve growth factor exon 1 promoter, fragment thereof, or modified form thereof.
  • nucleic acid according to 2 wherein the nucleic acid is human nerve growth factor exon 1 promoter 1 -1786, fragment thereof, or modified form thereof.
  • nucleic acid according to 2 wherein the nucleic acid is human nerve growth factor exon 1 promoter to 2274 - 2846, fragment thereof, or modified form thereof.
  • nucleic acid according to 1 wherein the nucleic acid is human nerve growth factor exon 3 promoter 1 - 1877, fragment thereof, or modified form thereof.
  • nucleic acid according to 1 , wherein the nucleic acid comprises a consensus binding motif from human nerve growth factor exon 1 promoter selected from 1 -1786,
  • human nerve growth factor exon 3 promoter 1 -1877 or modified form thereof.
  • nucleic acid according to 7 wherein the consensus binding motif comprises a CAAT box or TATA box.
  • the consensus binding motif is binding site for a ribosome.
  • nucleic acid according to 7, wherein the consensus binding motif is selected from the group consisting of NF-Ytk, NF-Y MCHII, AABS, ATF, Ad2MLP, EGR-1, ELP RS, GCN4 HIS3.1, GCN4 HIS4.3, GCN4 HIS4.4, GCRE, OBF H2B1, OBF histone, NF El .3, NF E1.6 and NF E1.5.
  • nucleic acid according to claim 6, wherein the consensus binding motif is selected from the group consisting of API, AP2, AP3, AP4, AP5, E4TF1, CTF/NF-1, NF-KB, TFIID, TFIIIA, p53, GM-CSF or NF IL-6.
  • nucleic acid according to 1 wherein the nucleic acid comprises an enhancer sequence, repressor sequence or consensus binding motif for a transcription activating factor.
  • nucleic acid according to 1 wherein the nucleic acid comprises a natural or a modified derivative of deoxyribonucleic acid or ribonucleic acid.
  • nucleic acid according to 12 wherein the nucleic acid comprises a phosphodiester, methylphosphonate, phosphoramidate, isopropyl phosphate triester, phosphorothioate, phosphothionate, phosphotriester or boranophosphate.
  • the present invention is also directed to manipulation of the human NGF exon 1 promoter, exon 3 promoter, fragment thereof, or modified form thereof, plasmids resulting from such manipulation and cells transformed or transfected with such plasmids and transgenic animals containing such plasmids.
  • the invention includes manipulation where exogenous promoters are inserted into human NGF exon 1 promoter or exon 3 promoter by, e.g., homologous recombination.
  • the invention also includes manipulation where all or part of a human exon 1 promoter or exon 3 promoter is replaced by a nonnaturally-occurring exogenous or otherwise endogenous DNA, which may be DNA from another gene, e.g., intron or exon of a gene other than NGF, from another chromosome, or a naturally-occurring variant of the human NGF exon 1 promoter or exon 3 promoter.
  • a nonnaturally-occurring exogenous or otherwise endogenous DNA which may be DNA from another gene, e.g., intron or exon of a gene other than NGF, from another chromosome, or a naturally-occurring variant of the human NGF exon 1 promoter or exon 3 promoter.
  • An example of an endogenous modification of human NGF exon 3 promoter would be e.g., part or all of human NGF exon 1 promoter replacing part or all of human NGF exon 3 promoter.
  • this manipulation includes where a nonnaturally occurring exogenous or otherwise endogenous DNA encoding consensus binding motif replaces, is inserted or is deleted from the naturally occurring consensus binding motif, e.g., where the consensus binding motif of AP3, which is the consensus binding motif for protein kinase C responsive element in human NGF exon 3, e.g., starting at +116, -1608 or +2472, is replaced with, for example, PRL, the prolactin gene regulatory control element at -159 of human NGF exon 3, deletion or alteration of a CAAT box or TATA box located, for example, in human NGF exon 3 promoter or a regulatory control element from another gene, or may even be a synthetically-derived control element based on a consensus sequence.
  • the consensus binding motif of AP3 which is the consensus binding motif for protein kinase C responsive element in human NGF exon 3
  • PRL the prolactin gene regulatory control element at -159 of human NGF exon 3
  • the invention is directed to insertion of regulatory elements, such as insertion of a CAAT box or TATA box in a non-naturally occurring site within human NGF exon 1 promoter or exon 3 promoter.
  • Such manipulation may be accomplished by, for example, homologous recombination or site directed mutagenesis.
  • the present invention is also directed to modifications of human NGF exon 1 promoter or exon 3 promoter which modify transcription of human NGF.
  • An example of such modification includes alteration of one or more lariat site in the human NGF exon 1 promoter or exon 3 promoter.
  • a lariat site is a loosely palindromic sequence which permits the DNA to loop back on itself.
  • Alteration of a lariat site may influence binding of transcription factors, even if the underlying consensus binding motif the transcription factor normally binds to is not altered.
  • Another example of such modification is alteration of a splice donor site or splice acceptor site.
  • the present invention is also directed to constructs resulting from such above manipulation, plasmids and vectors containing such constructs, and cells containing such constructs.
  • genetically altered cells suitable for autologous transplantation whereby human cells are manipulated to alter the naturally occurring NGF exon 1 promoter or exon 3 promoter to alter one, or more, naturally occurring consensus binding motif, add one, or more, non-naturally occurring consensus binding motif or delete, one or more, naturally occurring consensus binding motif, or other modifications of human NGF exon 1 promoter and/or exon 3 promoter.
  • the present invention is also directed to vectors comprising human NGF exon 1 promoter or exon 3 promoter, fragment thereof, or modifications thereof.
  • the present vectors include expression vectors, such as a vector comprising the human NGF exon 1 promoter or exon 3 promoter, fragment thereof or modification thereof, and a marker gene, such as a gene encoding a detectable protein or conferring an altered, or detectable, phenotype or genotype.
  • Especially preferred detectable proteins are reporter genes, and include luciferase, ⁇ - galactosidase, placental alkaline phosphatase and green fluorescent protein (GFP).
  • the present invention is also directed to reporter vectors, which comprise an insertional site for a gene of interest and the gene encoding neomycin resistance under control of a thymidine kinase promoter.
  • the present invention includes transformation vectors, such as a vector comprising the human NGF exon 1 promoter or exon 3 promoter, fragment thereof or modification thereof, and suitable for transfecting or transforming a suitable host cell.
  • suitable transformation vectors include plasmids pGL, pGEM and phages, such as gt 10 and gt 11.
  • Especially preferred vectors are defective viral vectors, including amplicons.
  • Defective viral vectors may result from one or more defective subgenomic viral particle(s) which contain an essential portion of the genome and require complementation of homologous "helper" virus for replication. Such defective viruses occur naturally and are also called defective interfering viruses (or Dl particles). Dl particles occur as RNA or DNA viruses, and have been identified in herpes viruses, including HSN, human cytomegalovirus, equine herpes virus. Especially preferred defective viral vectors of the present invention include amplicons comprising the human ⁇ GF exon 1 promoter or exon 3 promoter, fragment thereof or modification thereof. Preferred embodiments of vectors of the invention are as follows:
  • a vector comprising a nucleic acid human nerve growth factor exon 1 promoter 1 - 1786, 2274 - 2846, human nerve growth factor exon 3 promoter 1 - 1877, fragment thereof, or modified form thereof.
  • nucleic acid is nerve growth factor exon 1 promoter, fragment thereof, or modified form thereof.
  • nucleic acid is human nerve growth factor exon 1 promoter 1 - 1786, fragment thereof, or modified form thereof.
  • nucleic acid is human nerve growth factor exon 1 promoter to 2274 - 2846, fragment thereof, or modified form thereof.
  • nucleic acid is human nerve growth factor exon 3 promoter 1 - 1877, fragment thereof, or modified form thereof.
  • nucleic acid comprises a consensus binding motif from human nerve growth factor exon 1 promoter 1 - 1786, 2274 - 2846, human nerve growth factor exon 3 promoter 1 - 1877, fragment thereof, or modified form thereof.
  • nucleic acid comprises a consensus binding motif is selected from the group consisting of API, AP2, AP3, AP4, AP5, E4TF1, CTF/NF-1, NF-KB, TFIID, TFIIIA, p53, GM-CSF or NF IL-6.
  • nucleic acid comprises an enhancer sequence, repressor sequence or consensus binding motif for a transcription factor.
  • nucleic acid comprises a consensus binding motif of a transcription factor in an inflammatory pathway.
  • nucleic acid comprises a consensus binding motif of a transcription factor in a cell-death pathway.
  • nucleic acid comprises a consensus binding motif of a transcription factor in a tumorgenic pathway.
  • the vector according to 1 wherein the vector is an amplicon, transcription vector, expression vector, reporter vector, insertion vector, replacement vector, or mutagenesis vector.
  • the present invention is also direct to a novel vector designed to incorporate the human NGF exon 1 promoter, exon 3 promoter, fragment thereof, or modification thereof.
  • the vector comprises both a reporter gene and gene encoding antimetabolite resistance.
  • the present invention is also directed to cells comprising such vectors, methods of assaying compounds using the same, and methods for identifying a compound capable of modifying transcription of a nucleic acid. Specific embodiments of the present invention are as follows:
  • a vector comprising pGL3-neo.
  • the vector according to 1, comprising a promoter sequence greater than 2 kilobases.
  • the vector according to 1 comprising a nucleic acid encoding human nerve growth factor exon 1 promoter 1 - 1786, 2274 - 2846, human nerve growth factor exon 3 promoter 1 1877, fragment thereof, or modified form thereof.
  • nucleic acid comprises human nerve growth factor exon 1 promoter 1 - 1786, 2274 - 2846, fragment thereof, or modified form thereof.
  • nucleic acid comprises human nerve growth factor exon 1 promoter 1 - 1786, fragment thereof, or modified form thereof.
  • nucleic acid comprises human nerve growth factor exon 1 promoter 2274 - 2846, fragment thereof, or modified form thereof.
  • nucleic acid comprises human nerve growth factor exon 3 promoter 1 - 1877, fragment thereof, or modified form thereof.
  • a cell comprising a vector according to 1.
  • a cell according to 11 wherein the cell is a human or primate cell.
  • An assay comprising a cell according to 10.
  • a method for identifying a compound capable of modifying transcription of a nucleic acid comprising contacting a compound with a cell according to 1.
  • the present invention may also be used in recombinant technology to produce proteins. Therefore, the present invention is directed to vectors wherein the human NGF exon
  • the invention is also directed to methods of producing protein using the human NGF exon 1 promoter, or exon 3 promoter, fragment thereof, or modified form thereof.
  • Preferred embodiments of the invention include the following:
  • a method of producing a protein comprising expressing a vector comprising a human nerve growth factor exon 1 promoter 1 - 1786, 2274 - 2846, human nerve growth factor exon 3 promoter 1 - 1877, fragment thereof, or modified form thereof operably linked to a gene encoding a protein.
  • the promoter comprises a human nerve growth factor exon 1 promoter 1 - 1786, 2274 - 2846, fragment thereof, or modified form thereof.
  • the promoter comprises a human nerve growth factor exon 1 promoter selected from 1 - 1786, fragment thereof, or modified form thereof.
  • the promoter comprises a human nerve growth factor exon 1 promoter selected from 2274 - 2846, fragment thereof, or modified form thereof.
  • the promoter comprises a human nerve growth factor exon 3 promoter 1 - 1877, fragment thereof, or modified form thereof.
  • the vector comprises a consensus binding motif from human nerve growth factor exon 1 promoter 1 - 1786, 2274 - 2846, human nerve growth factor exon 3 promoter 1 - 1877, fragment thereof, or modified form thereof.
  • the selectable protein confers antimicrobial resistance.
  • the antimicrobial resistance is to neomycin, sulfonamide, penicillin, cephalosporin, aminoglycoside, tetracyclin, or modified forms thereof.
  • the present invention also includes oligonucleotides encoding human NGF exon 1 promoter or exon 3 promoter, fragment thereof, or modified form thereof.
  • Preferred oligonucleotides are antisense oligonucleotides to a fragment of either human NGF exon 1 promoter or exon 3 promoter. More preferred antisense oligonucleotides are to all or part of a consensus binding motif within either human NGF exon 1 promoter or exon 3 promoter.
  • Preferred oligonucleotides are about six to about one hundred bases long.
  • Preferred antisense oligonucleotides are six to one hundred bases long, more preferred antisense oligonucleotides are about six to about fifty bases long, and even more preferred antisense oligonucleotides are about ten to about twenty five bases long.
  • Especially preferred antisense oligonucleotides are about fifteen bases long.
  • Nucleic acid of the present invention may contain naturally occurring nucleotides or analogs thereof.
  • Preferred naturally-occurring nucleotides are either deoxyribonucleic acid or ribonucleic acid.
  • Preferred analogs of naturally-occurring nucleotides are modified phosphotriesters, bases or sugars. Especially preferred are phosphodiesters, methylphosphonates, phosphoramidates, isopropyl phosphate triesters, phosphorothioates, phosphothionates, phosphotriesters or bo
  • the present invention includes methods of modifying regulation of human nerve growth factor by administration of an oligonucleotide encoding human NGF exon 1 promoter or exon 3 promoter, fragment thereof, or modified form thereof.
  • a preferred method is by administration of an antisense oligonucleotide of human NGF promoter of exon 1 or 3.
  • An especially preferred method is by administration of an antisense oligonucleotide to a consensus binding motif of human NGF exon 1 promoter or exon 3 promoter.
  • the present invention is also directed to methods for gene therapy involving altering naturally occurring transcriptional control of human NGF.
  • the present invention includes methods of transfecting cells and the transformed cells.
  • a method of transferring a nucleic acid to a cell comprising administering to the cell a nucleic acid encoding human nerve growth factor exon 1 promoter 1 - 1786, 2274 - 2846, human nerve growth factor exon 3 promoter 1 - 1877, fragment thereof, or modified form thereof.
  • nucleic acid comprises a consensus binding motif from human nerve growth factor exon 1 promoter selected from 1 - 1786, 2274 - 2846, human nerve growth factor exon 3 promoter 1 - 1877, fragment thereof, or modified form thereof.
  • nucleic acid comprises deoxyribonucleic acid, ribonucleic acid, or modified form thereof.
  • nucleic acid comprises a modified form of nucleic acid.
  • nucleic acid comprises a phosphodiester, methylphosphonate, phosphoramidate, isopropyl phosphate triester, phosphorothioate, phosphothionate, phosphotriester or boranophosphate.
  • a transformed cell comprising a nucleic acid encoding human nerve growth factor exon 1 promoter 1 - 1786, 2274 - 2846, human nerve growth factor exon 3 promoter 1 - 1877, fragment thereof, or modified form thereof.
  • nucleic acid comprises nerve growth factor exon 1 promoter 1 - 1786, 2274 - 2846, fragment thereof, or modified form thereof.
  • nucleic acid comprises human nerve growth factor exon 1 promoter 1 - 1786, fragment thereof, or modified form thereof.
  • nucleic acid comprises human nerve growth factor exon 1 promoter to 2274 - 2846, fragment thereof, or modified form thereof.
  • nucleic acid is human nerve growth factor exon 3 promoter 1 - 1877, fragment thereof, or modified form thereof.
  • nucleic acid comprises a consensus binding motif from human nerve growth factor exon 1 promoter 1 - 1786, 2274 - 2846, human nerve growth factor exon 3 promoter 1 - 1877, fragment thereof, or modified form thereof.
  • the present invention is also directed to methods of making animal models useful to study NGF regulation and to the resulting animals. Embodiments of such methods and resulting animals are as follows:
  • a method of transferring a nucleic acid into an animal comprising administering to the animal a nucleic acid encoding human nerve growth factor exon 1 promoter 1 - 1786, 2274 - 2846, human nerve growth factor exon 3 promoter 1 - 1877, fragment thereof, or modified form thereof.
  • nucleic acid comprises a consensus binding motif from human nerve growth factor exon 1 promoter 1 - 1786, 2274 - 2846, human nerve growth factor exon 3 promoter 1 - 1877, fragment thereof, or modified form thereof.
  • nucleic acid comprises deoxyribonucleic acid, ribonucleic acid, or modified forms thereof.
  • nucleic acid comprises a modified form of nucleic acid.
  • nucleic acid comprises a phosphodiester, methylphosphonate, phosphoramidate, isopropyl phosphate triester, phosphorothioate, phosphothionate, phosphotriester or boranophosphate.
  • the vector is adenovirus, adenoassociated virus, retrovirus, herpes virus, or modifications thereof.
  • the vector is an amplicon.
  • the present invention includes animal models with human NGF exon 1 promoter or exon 3 promoter, fragment thereof, or modification thereof. Such modifications may be deletion, alteration, or inclusion of one or more consensus binding motif(s) of the endogenous NGF promoter in exon 1 and/or exon 3 of that animal which correspond to a consensus binding motif in the human NGF promoter exon 1 or exon 3. Included are animal models which are transgenic animals containing human NGF promoter of exon 1 or 3, or both exons 1 and 3, or hybrids thereof. Especially preferred animal models include animal models comprising amplicon-based NGF promoter of either exon 1 or exon 3, or both, or modifications thereof.
  • Amplicons of the present invention differ slightly from previous examples of amplicons, where the amplicon is used to express a gene of interest.
  • an amplicon is a vector where the endogenous viral promoter is substituted with all or part of either human NGF promoter of exon 1 or 3, or both exons 1 and 3, or hybrids thereof, and optionally include all or part of NGF gene exons.
  • Embodiments of the animal models of the present invention are as follows: 1 A nonhuman animal comprising human nerve growth factor exon 1 promoter 1 - 1786, 2274 - 2846, human nerve growth factor exon 3 promoter 1 - 1877, fragment thereof, or modified form thereof.
  • nucleic acid is human nerve growth factor exon
  • nucleic acid is human nerve growth factor exon
  • nucleic acid comprises a consensus binding motif from human nerve growth factor exon 1 promoter selected from 1 - 1786, 2274 - 2846 or human nerve growth factor exon 3 promoter 1 - 1877, or modified therefrom.
  • nucleic acid comprises a consensus binding motif is selected from the group consisting of API, AP2, AP3, AP4, AP5, E4TF1, CTF/NF-1, NF-KB, TFIID, TFIIIA, p53, GM-CSF or NF IL-6.
  • nucleic acid comprises an enhancer sequence, repressor sequence or consensus binding motif for a transcription activating factor.
  • nucleic acid comprises a natural or a modified derivative of deoxyribonucleic acid or ribonucleic acid.
  • the invention includes methods and assays for a compound capable of modifying human nerve growth factor regulation.
  • a preferred embodiment of a method is contacting a compound with human NGF exon 1 promoter, exon 3 promoter, fragment thereof, or modification thereof.
  • a more preferred embodiment of the present invention includes a vector comprising a modified form of human NGF exon 1 promoter or exon 3 promoter, fragment thereof, or modification thereof, such as one comprising a deletion of one or more consensus binding motif or other modification, such as a modified lariat site, altered splice donor site or splice acceptor site, or combinations thereof, cells containing such vectors comprising such vectors and assays using such cells.
  • Embodiments of assay methods are as follows:
  • a method of identifying a compound capable of modifying human nerve growth factor regulation comprising administering a compound to a cell, wherein the cell comprises a vector which comprises a human nerve growth factor exon 1 promoter 1 - 1786, 2274 - 2846, human nerve growth factor exon 3 promoter 1 - 1877, fragment thereof, or modified form thereof.
  • the vector comprises a human nerve growth factor exon 1 promoter 1 - 1786, 2274 - 2846, fragment thereof, or modified form thereof.
  • the vector ' comprises a consensus binding motif from human nerve growth factor exon 1 promoter 1 - 1786, 2274 - 2846, human nerve growth factor exon 3 promoter 1 - 1877, fragment thereof, or modified form thereof.
  • the selectable protein confers antimicrobial resistance.
  • the antimicrobial resistance is to neomycin, sulfonamide, penicillin, cephalosporin, aminoglycoside, tetracyclin, or modified forms thereof.
  • the present invention is also directed to a method for identifying compounds capable of modifying transcription of human NGF.
  • Preferred embodiments of the invention are directed to a method of characterizing a compound capable of modifying initiation of transcription of human nerve growth factor exon 1 promoter or human nerve growth factor exon 3 promoter. More preferred embodiments of the invention are as follows:
  • a method for identifying a compound capable of modifying transcription of human nerve growth factor exon 1 promoter or human nerve growth factor exon 3 promoter comprising contacting a cell comprising a nucleic acid encoding human nerve growth factor exon 1 promoter 1 - 1786, 2274 - 2846, human nerve growth factor exon 3 promoter 1 - 1877, fragment thereof, or modification thereof, with a compound and detecting modification of initiation of transcription.
  • reporter gene is luciferace, green fluorescent protein, modified form thereof, ⁇ -galactosidase, or placental alkaline phosphatase.
  • the present invention is also directed to a method for characterizing compounds capable of modifying transcription of human NGF.
  • Preferred embodiments of the invention are directed to a method of characterizing a compound capable of modifying initiation of transcription of human nerve growth factor exon 1 promoter or human nerve growth factor exon 3 promoter. More preferred embodiments of the invention are as follows:
  • a method of characterizing a compound capable of modifying transcription of human nerve growth factor comprising contacting a cell comprising a nucleic acid encoding human nerve growth factor exon 1 promoter 1 - 1786, 2274 - 2846, human nerve growth factor exon 3 promoter 1 - 1877, fragment thereof, or modification thereof, with a compound and detecting modification of transcription.
  • reporter gene is luciferace, green fluorescent protein, modified form thereof, ⁇ -galactosidase, or placental alkaline phosphatase.
  • the present invention is also directed to a compound capable of modifying transcription of human NGF.
  • Preferred embodiments of the invention are directed to a compound capable of modifying initiation of transcription of human nerve growth factor exon 1 promoter or human nerve growth factor exon 3 promoter. More preferred embodiments of the invention are as follows: 1. A compound capable of binding to a human nerve growth factor exon 1 promoter 1 -
  • DNA for the human nerve growth factor exon 3 clones was originally identified by PCR screening a human PI genomic library (clone 0095-B8, Genome Systems). A -6600 bp fragment containing exon 3 was cloned into a pBS SK+ vector to yield the plasmid identified as pBSEx3. A 4329 bp fragment was isolated from the insert in pBSEx3 and subcloned into a pGL2 enhancer vector (Promega) and a pGL3 basic vector (Promega) to yield clones identified as pGL2Ex3 and pGL3Ex3, respectively. The pGL2Ex3 was transfected into mouse L929 cells and the pGL3Ex3 vector was transfected into human UC11 cells to generate the data in Table 1.
  • DNA for the human nerve growth factor exon 1 clones was originally identified by PCR screening a human PI genomic library (clone 1226-G9, Genome Systems). A -14,000 bp fragment containing exon 1 was cloned into a pBS SK+ vector to yield the plasmid identified as pBSExl. Two overlapping fragments were isolated from the insert in pBSExl and subcloned into a pGL3neo vector. The largest construct containing human nerve growth factor exon 1 is 2846 bp and is identified as pNElKS.
  • the second subclone from pBSExl contains the same 5' end as pNElKS and is truncated on the 3' end in exon 1, resulting in an insert that is 2234 bp.
  • pNElKS and pNEIKE were transfected into mouse L929 cells and human UC11 cells to generate the data in Table 1.
  • Oligonucleotides used to screen a genomic PI library (Genome Systems, St. Louis, MO) for clones containing the area of interest as well as internal oligonucleotides used in restriction digestion analysis to locate appropriately sized regions to subclone are provided in Table 2.
  • Species indicates the species to which the sequence is homologous. Mouse oligonucleotides were found to be cross reactive to human DNA.
  • Primers #4 and #5 (in Table 2) were used to amplify sequence from human NGF exon 1 and primers #1 and #2 were used for exon 3 identification. ' Each oligonucleotide (400 nM) was used in separate reactions for exon 1 and exon 3. Template for these reactions was 1/40 the DNA from each PI mini -prep described below. The reaction also contained 10 mM Tris-HCl pH 8.3, 50 mM KCl, 3 mM MgCl 2 , 250 ⁇ M each dATP, dCTP, dGTP, dTTP, and 2.5 U Taq
  • DNA polymerase Perkin Elmer, Norwalk, CT
  • Amplification was carried out using a Perkin Elmer 460 thermocycler programmed to 95°C for 5 min and then cycled through 95°C, 30 s; 60°C, 30 s; 72°C, 1 min for 35 cycles.
  • Control reactions were set up containing 500 ng of human genomic DNA as a positive template for the PCR reaction.
  • the oligonucleotide #4 to human NGF exon 1, and #3 to exon 3B were end labeled to locate fragments containing exon 1 or 3 in blots of restriction digests and subcloned DNA.
  • Exon 1 Promoter Isolation Two primers, #4 and #5 (Table 2), designed to amplify human NGF exon 1, identified three genomic clones, all of which contained exon 1. One of these clones, Clone #1226-G9, was digested with Kpn I to yield a 14 kb band which was ligated into the Kpn I site of pBS II SK+.
  • pNEIKE is a truncated portion of human nerve growth factor exon 1 promoter 1 to 2234 bp insert
  • pNElKS which contains a 2846 bp insert of human nerve growth factor exon 1 promoter of 1 to 2846 SEQUENCE ID NO. 6 of the following sequence in Table 3.
  • GGTACCACTG CCAGCACACA GTGCCTGGCA TATGGTAGGC TCTCAATCAA 50 TAATCTTTGG AGTATTTTTG TGTTTGTTGT TTACATGTTC TTATTTACTC 100 AAGATCCTTG AAGTCCAGGG ACAGAAATAG AGGTAGTTAG GGGCAGAAAG 150 GAGCTCTTAT TAAATCAACA TGTGCAAGAA GAATATGACC AACAATTTAG 200 GGGGTGAGGA TGGAGCATAT AAGCAAACTT ATAATCTGCT TACATCACTT 250 AAAGTTTCCC CCTTACATAC CACATGGAAA AGAACCACAA GTGTCCCAAA 300 TCCTTTTGTC CTTCTGAATG ATGCCACAAG AACACATACA AATGCTCTGC 350 ATTCAACAAC CAAATTCTCT GTTATTCTAA AAGTTTAATT TCATACCCAA 400 ATTCTCAGGC AGCTATTATG TAAGGCTTGG GGCTAGTGCT TTCCAAACAA 450 GTTTATACAT
  • the human PI library was screened with cross-reactive mouse exon 3 primers, #1 and #2 (Table 2).
  • An Asp718/Pvu 1 digestion of clone #0095-B8 yielded an 6600 bp band containing exon 3.
  • This fragment was subcloned into the Asp718 site of pBS SK+, and the resulting plasmid was referred to as pBSEx3.
  • This clone was verified by restriction mapping and was used to generate sufficient DNA for subcloning into the luciferase expression vectors pGL2 enhancer and pGL3 basic.
  • the pGL2Ex3 plasmid was digested with Hind III and the same insert subcloned into the Hind III site of pGL3 basic vector to yield the plasmid referred to as pGL3Ex3 used for the UC11 stable cell line.
  • Stable transfectants of UC11 or L929 cells containing the pGL3Ex3 plasmid or the pGL2Ex3 plasmid and the G418 resistant plasmid pCDNA3, were selected on the basis of their ability to survive in media containing 600 ⁇ g/ml G418 and express luciferase activity. From these co- transfections, 34% and 36% of clones screened showed luciferase activity in L929 and UC11 cells, respectively, indicating incorporation of the exon 3 promoter region. One cell line from each transfection was selected for further evaluation and a number of assays were conducted to characterize the cell lines and test functionality of the NGF promoter region in these cells.
  • a luciferase-based reporter plasmid was used to investigate the nerve growth factor exon 1 and exon 3 promoters.
  • the thymidine kinase promoter and neomycin resistance gene excised from pMClneo (Stratagene, LaJolla, CA) using Xho I, were cloned into the Sal I cut plasmid pGL3 -basic (Promega, Madison, WI).
  • the resulting vector was designated "pGL3-neo" and is 5960 bp.
  • One advantage of this vector is the dual incorporation of a selectable marker, here, neomycin resistance, and a reporter gene, here the luciferase gene.
  • This vector avoids the necessity of co-transfection, and is stable over multiple passages and the transfected cell line maintains a high level of desired protein expression, here luciferase. Thus, this vector is particularly desirable for high-throughput assays.
  • Another advantage is the small size, which permits relatively large insertions of the promoter or other control elements of interest.
  • Still another advantage of this vector is that incorporation of the selectable gene and promoter, here tk-neo, affects only one of the otherwise unique restriction sites, Mlu 1, in the pGL3-basic vector. Thus, the remaining unique restriction endonuclease sites, Kpn 1, Sac I, Nhe I, Sma I, Xho I, Bgl II, and Hind III, are unaffected.
  • the complete sequence of pGL3-neo SEQUENCE ID NO. 14 is
  • Glycerol stocks of bacterial cells containing P ] genomic DNA were used to inoculate Luria Broth (LB) containing 25 ⁇ g/ml kanamycin. The cultures were grown overnight at 37°C and mini preps prepared by a modified alkaline lysis method as recommended by the manufacturer. DNA was used within 24 hours for restriction analysis or stored in small aliquots at -20°C to avoid repeated thawing and freezing. For DNA subcloning, 20 mis of overnight culture were processed as 1.5 ml aliquots, pooled, digested with the appropriate restriction enzymes and size fractionated on a gel.
  • End-labeling of oligonucleotides as probes for exon 3 was performed using ⁇ [32p] ATP (Amersham, Arlington Heights, IL), specific activity >5000 Ci/mmol, in a 2:1 pmol ratio with oligonucleotide.
  • the oligonucleotide was denatured by placing in boiling water for 2 minutes, then mixed with the radioactive ATP and dried in a vacuum desiccator. The mix was resuspended in 50 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 5 mM DTT, 10 units T4 polynucleotide kinase (PNK)
  • Hybridization of exon 3 blots was carried out by first pre-hybridizing blots in 6X SSC, 5X Denhart's, 100 ⁇ g/ml salmon sperm DNA, 0.5% SDS, 0.2 M NaPO 4 (pH 7.0), at 50°C for 3-6 hours.
  • radioactive blots were placed on a phosphorimager screen for 5-24 hours and scanned by a Molecular Dynamics SF phosphorimager using ImageQuant software analysis (Molecular Dynamics, Sunnyvale, CA). ECL screened blots were placed on film (Hyperfilm ECL, Amersham) for 10 to 30 minutes. '
  • Vector DNA (5 ⁇ g, pBS SK+ (Stratagene, La Jolla, CA), pGL2 Enhancer, pGL3 basic (Promega, Madison, WI), or pGL3 neo) was digested with the appropriate restriction endonuclease and incubated with 25-50 units calf intestinal alkaline phosphatase (Gibco/BRL, Gaithersburg, MD) to remove the 5' phosphate group and reduce self-ligation. The reaction was carried out in 50 mM Tris-HCl (pH 8.5), 0.1 mM EDTA at 37°C for 30 minutes.
  • the DNA was run on a 1% agarose gel (Ultrapure agarose, Gibco/BRL) at 80-100 volts, and the linearized band excised and purified with Geneclean. Both insert and vector DNA were diluted to -50 ng/ ⁇ l and ligated in a 3:1 ratio for 15-18 hours at 14°C in 50 mM Tris-HCl (pH7.6), 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 5% polyethylene glycol-8000 with 0.5 units T4 ligase.
  • Transformation was carried out by mixing 50 ⁇ l of maximum efficiency DH5 ⁇ cells (Gibco/BRL) with 2 ⁇ l of undiluted ligation reaction mix on ice for 30 minutes. The cells were heat shocked 40 sec at 42°C, returned to ice for 2 min and 950 ⁇ l SOC media was added to begin recovery. The cells were shaken at 225 rpm in SOC at 37°C for 1 hour and 200 ⁇ l of this suspension was spread on an agar plate containing 50 ⁇ g/ml ampicillin. Agar plates were incubated at 37°C overnight for growth of colonies. Clones containing the appropriate plasmid insert were identified by restriction analysis and confirmed by sequencing.
  • L929 mouse fibroblast cells (ATCC, Rockville, MD) were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% horse serum, penicillin (50 ⁇ g/ml), streptomycin (50 ⁇ g/ml), neomycin (100 ⁇ g/ml), and glutamine (1 mM). Cells were maintained at 37°C in 5% CO2, fed every 3-4 days and passaged once per week.
  • DMEM Dulbecco's modified Eagle's medium
  • serum free media When serum free media was used before luciferase assays, it contained DMEM:Ham's F12 (3:1), insulin (5 ⁇ g/ml), transferring (5 ⁇ g/ml), sodium selenite (5 ng/ml), penicillin (50 ⁇ g/ml), streptomycin (50 ⁇ g/ml), neomycin (100 ⁇ g/ml) and glutamine (1 mM).
  • UC11 human astrocytoma cells (Liwnicz, et. al. 1986) were grown in RPMI 1640 containing 10% ⁇ fetal bovine serum, 20 mM HEPES, penicillin (50 ⁇ g/ml), streptomycin (50 ⁇ g/ml), neomycin (100 ⁇ g/ml), and glutamine (1 mM). Cells were maintained at 37°C in 5% CO2, fed every 3-4 days and passaged once per week.
  • serum free media When serum free media was used before luciferase assays, it contained RPM Ham's F12 (3:1), 20 mM HEPES, insulin (5 ⁇ g/ml), transferring (5 ⁇ g/ml), sodium selenite (5 ng/ml), penicillin (50 ⁇ g/ml), streptomycin (50 ⁇ g/ml), neomycin (100 ⁇ g/ml) and glutamine (1 mM).
  • Exon 1 clones were prepared by electroporation of 10 ⁇ g pNEIKE or pNElKS DNA into 5 x 10 L929 or UC11 cells.
  • the exon 3 clones required co-transfection with pCDNA3 (Invitrogen, San Diego, CA) containing the neomycin resistance gene which confers G418 resistance allowing selection of transfectants.
  • pCDNA3 Invitrogen, San Diego, CA
  • L929 cells were electroporated with 10 ⁇ g pGL2Ex3 DNA and 1 ⁇ g pCDNA3 and UC11 cells were electroporated with 10 ⁇ g pGL3Ex3 DNA and 1 ⁇ g pCDNA3.
  • HBSS Hank's Balanced Salt Solution
  • Cells were plated at 5,000 cells/well in 96 well dishes in serum containing media described above. The next day cells were washed twice and incubated for an additional 48-56 hours in serum free media. Cells were treated with 1 ⁇ M PMA, 10 nM calcitriol or 10% horse serum and luciferase activity was determined 18 hours later using a Promega kit (catalog #E1500).
  • media was aspirated and cells were lysed in 200 ⁇ l cell lysis buffer (containing 25 mM Tris-phosphate, pH 7.8, 2 mM DTT, 2 mM l,2-diaminocyclohexane-N,N,N r N'-tetraacetic acid, 10% glycerol, 1% triton X-100).
  • 100 ⁇ l cell/buffer solution was transferred to a white Dynatech microlite 2, 96 well dish. Luciferase activity was detected in a MicroLumat LB 96 P lumihometer (Wallac Inc, Gaithersburg, MD) for 10 seconds following automatic injection of 100 ⁇ l 470 ⁇ M luciferin.
  • Consensus binding motifs in the sequences human nerve growth factor exon 1 and exon 3 promoters were determined using Mac Vector, Ver 4.0, (IBI, Inc, NewHaven, CT) . Putative consensus sequences were scanned for relatively high fidelity to the consensus binding motif and are preferred consensus binding motifs in human nerve growth factor exon 1 and exon 3 promoters. Table 6 provides a partial list of consensus binding motifs. TABLE 6
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • CCGGGCAGAC CAGGGAGGCA CTGGCGAAGG GCAACGCGCGCG GGGGCAGGGC GGAGAGGTGA 1860
  • MOLECULE TYPE DNA (genomic)
  • CAGGCTG 487 INFORMATION FOR SEQ ID NO: 8:
  • MOLECULE TYPE DNA (genomic)
  • CTCCCTGTTA CTCTCCTCAC ACCTACTTCT CCTCTGTGGC ATCCATACAG GGTAGGGGTC 1080
  • MOLECULE TYPE DNA (genomic)
  • ATGTACTGAA CAATTTCTAT TTTGATGCCA GATTAGGGAT CTGCTGGGGC AAGACTTTGG 3780
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • GGACAAACCA CAACTAGAAT GCAGTGAAAA AAATGCTTTA TTTGTGAAAT TTGTGATGCT 1860
  • ATAAAGATAC CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG TTCCGACCCT 3600
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO : 34 :
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO: 71: ATTTGCAT 8
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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EP98901211A 1997-02-06 1998-01-12 Promotoren von exon 1 und exon 3 des menschlichen nervenwachstumsfaktors. Withdrawn EP0977778A2 (de)

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