Genomic DNA fragments containing regulatory and coding sequences for the B2-subunit of the neuronal nicotinic acetylcholine receptor and transgenic animals made using these fragments or mutated fragments

Abstract

Several genes encoding subunits of the neuronal nicotinic acetylcholine receptors have been cloned and regulatory elements involved in the transcription of the ∝:2 and ∝:7-subunit genes have been described. Yet, the detailed mechanisms governing the neuron-specific transcription and the spatio-temporal expression pattern of these genes remain largely uninvestigated. The β2-subunit is the most widely expressed neuronal nicotinic receptors subunit in the nervous system. We have studied the structural and regulatory properties of the 5′ sequence of this gene. A fragment of 1163 bp of upstream sequence is sufficient to drive the cell-specific transcription of a reporter gene in both transient transfection assays and in transgenic mice. Deletion analysis and site-directed mutagenesis of this promoter reveal two negative and one positive element. The positively acting sequence includes one functional E-box. One of the repressor elements is located in the transcribed region and is the NRSE/RE1 sequence already described in promoters of neuronal genes.

1 - 19 . (canceled) 
     
     
         20 . A method for isolating the genomic DNA clone for the β2-subunit of the mouse neuronal nicotinic acetylcholine receptor comprising providing a mouse genomic DNA library and hybridizing under suitable conditions a DNA probe encoding the β2-subunit of neuronal nicotinic acetylcholine receptor from another mammalian species. 
     
     
         21 - 23 . (canceled) 
     
     
         24 . A nucleic acid probe consisting essentially of the sequence set forth in  FIG. 1  (SEQ ID NO. 22), a sequence hybridizing to the sequence set forth in  FIG. 1  under stringent conditions, or a fragment thereof. 
     
     
         25 - 27 . (canceled) 
     
     
         28 . A method for isolating neurons from non-human tissue comprising:
 providing a transgenic, non-human mammal all of whose germ cells and somatic cells contain a DNA introduced into the mammal or an ancestor of the mammal at an embryonic stage, wherein the DNA contains a promoter of the β2-subunit of neuronal nicotinic acetylcholine receptor consisting essentially of the sequence set forth in  FIG. 1  (SEQ ID NO. 22), a sequence hybridizing to the sequence set forth in  FIG. 1  under stringent conditions, or a fragment thereof, wherein the DNA is operatively linked to a nucleotide sequence encoding a protein, polypeptide, or peptide, and wherein the encoded protein, polypeptide, or peptide is a reporter gene,   identifying the neurons which express the reporter gene, and   separating the neurons that express the reporter gene from other cells.   
     
     
         29 . A method for targeting the expression of a desired polypeptide, protein or peptide product to neurons in a non-human, transgenic mammal comprising replacing the gene for the β2-subunit of neuronal nicotinic acetylcholine receptor with the DNA encoding the desired product in the genome of the mammal by homologous recombination, where said product is encoded by a DNA sequence. 
     
     
         30 . A transgenic, non-human mammal all of whose germ cells and somatic cells contain DNA introduced into the mammal or an ancestor of the mammal at an embryonic stage, wherein the DNA contains a promoter of the β2-subunit of neuronal nicotinic acetylcholine receptor consisting essentially of the sequence set forth in  FIG. 1  (SEQ ID NO. 22), a sequence hybridizing to the sequence set forth in  FIG. 1  under stringent conditions, or a fragment thereof, wherein the DNA is operatively linked to a nucleotide sequence encoding a protein, polypeptide, or peptide, and wherein the DNA has been mutated by point mutation, deletion, insertion or other means whereby the expression of the protein, polypeptide, or peptide has been altered as measured by biochemical assay or behavioral assay of a transgenic mammal. 
     
     
         31 . A cell line produced from the mammal as claimed in  claim 30 . 
     
     
         32 - 34 . (canceled) 
     
     
         35 . An isolated DNA consisting essentially of the sequence set forth in  FIG. 1  (SEQ ID NO. 22), a sequence hybridizing to the sequence set forth in  FIG. 1  under stringent conditions, or a fragment thereof, wherein the DNA is operatively linked to a nonhomologous nucleotide sequence encoding a protein, polypeptide, or peptide, wherein the DNA comprises sequences sufficient to promote transcription of the operatively linked nucleotide sequence in neuronal cells. 
     
     
         36 - 39 . (canceled) 
     
     
         40 . A nucleic acid probe consisting essentially of the sequence encoding at least one exon of the β2-subunit of neuronal nicotinic acetylcholine receptor. 
     
     
         41 . A method of directing expression of a peptide in a cell, comprising incorporating into the cell a DNA operatively linked to a nucleotide sequence encoding a peptide, polypeptide, or protein, wherein the DNA is selected from the group consisting of:
 (A) the sequence from about nucleotide −1125 to about nucleotide +38 as set forth in  FIG. 1  (SEQ ID NO. 22);   (B) a sequence having promoter activity, which hybridizes to DNA complementary to said sequence (A) under stringent conditions, wherein said stringent conditions comprise a temperature of about 65° C. and an SSC buffer concentration of about 0.1×SSC;   (C) the sequence from about nucleotide −968 to about nucleotide +38 as set forth in  FIG. 1 ;   (D) the sequence from about nucleotide −824 to about nucleotide +38 as set forth in  FIG. 1 ;   (E) the sequence from about nucleotide −245 to about nucleotide +38 as set forth in  FIG. 1 ; and   expressing the polypeptide.   
     
     
         42 . The method of  claim 41 , wherein the cell is a neuronal cell. 
     
     
         43 . The method of  claim 41 , wherein the peptide, polypeptide, or protein is encoded by a reporter gene. 
     
     
         44 . The method of  claim 43 , wherein the reporter gene encodes β-galactosidase or Luciferase. 
     
     
         45 . The method of  claim 41 , wherein the peptide, polypeptide, or protein is a toxin, growth factor, neuropeptide, tumorigenic protein, oncogenic protein, or immortalizing protein. 
     
     
         46 . A method of identifying a cell expressing a peptide, comprising
 incorporating into the cell a DNA operatively linked to a nucleotide sequence encoding a peptide, wherein the DNA is selected from the group consisting of:
 (A) the sequence from about nucleotide −1125 to about nucleotide +38 as set forth in  FIG. 1  (SEQ ID NO. 22); 
 (B) a sequence having promoter activity, which hybridizes to DNA complementary to said sequence (A) under stringent conditions, wherein said stringent conditions comprise a temperature of about 65° C. and an SSC buffer concentration of about 0.1×SSC; 
 (C) the sequence from about nucleotide −968 to about nucleotide +38 as set forth in  FIG. 1 ; 
 (D) the sequence from about nucleotide −824 to about nucleotide +38 as set forth in  FIG. 1 ; 
 (E) the sequence from about nucleotide −245 to about nucleotide +38 as set forth in  FIG. 1 ; 
   expressing the polypeptide; and   identifying the cell expressing the polypeptide.   
     
     
         47 . The method of  claim 46 , wherein the cell is a neuronal cell. 
     
     
         48 . The method of  claim 46 , wherein the peptide is encoded by a reporter gene. 
     
     
         49 . The method of  claim 48 , wherein the reporter gene encodes β-galactosidase or Luciferase. 
     
     
         50 . The method of  claim 46 , wherein the peptide is a toxin, growth factor, neuropeptide, tumorigenic protein, oncogenic protein, or immortalizing protein. 
     
     
         51 . A method of isolating a cell expressing a peptide, comprising
 incorporating into the cell a DNA operatively linked to a nucleotide sequence encoding a peptide, wherein the DNA is selected from the group consisting of:
 (A) the sequence from about nucleotide −1125 to about nucleotide +38 as set forth in  FIG. 1  (SEQ ID NO. 22); 
 (B) a sequence having promoter activity, which hybridizes to DNA complementary to said sequence (A) under stringent conditions, wherein said stringent conditions comprise a temperature of about 65° C. and an SSC buffer concentration of about 0.1×SSC; 
 (C) the sequence from about nucleotide −968 to about nucleotide +38 as set forth in  FIG. 1 ; 
 (D) the sequence from about nucleotide −824 to about nucleotide +38 as set forth in  FIG. 1 ; 
 (E) the sequence from about nucleotide −245 to about nucleotide +38 as set forth in  FIG. 1 ; 
   expressing the polypeptide; and   isolating the cell expressing the polypeptide.   
     
     
         52 . The method of  claim 51 , wherein the cell is a neuronal cell. 
     
     
         53 . The method of  claim 51 , wherein the peptide is encoded by a reporter gene. 
     
     
         54 . The method of  claim 52 , wherein the reporter gene encodes β-galactosidase or Luciferase. 
     
     
         55 . The method of  claim 50 , wherein the peptide is encoded by a toxin, growth factor, neuropeptide, tumorigenic protein, oncogenic protein, or immortalizing protein.