Genomic DNA fragments containing regulatory and coding sequence for the beta2-subunit of the neuronal nicotinic acetylcholine receptor and transgenic animals made using these fragments or mutated fragments

• US 20050003407A1
• Filed: 05/12/2004
• Published: 01/06/2005
• Est. Priority Date: 12/14/1994
• Status: Active Grant

• Current Assignee: Institut Pasteur
• Original Assignee: Institut Pasteur
• Inventors: Picciotto, Marina, Bessis, Alain, Changeux, Jean-Pierre
• Granted Patent: US 7,294,463 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/0312 Animal model for Alzheimer's disease
• CPC Class Codes: A01K 2267/0318 Animal model for neurodegenerative disease, e.g. non- Alzheimer's
• CPC Class Codes: A01K 2267/0331 Animal model for proliferative diseases
• CPC Class Codes: A01K 2267/0356 Animal model for processes and diseases of the central nervous system, e.g. stress, learning, schizophrenia, pain, epilepsy
• CPC Class Codes: A01K 2267/0393 Animal model comprising a reporter system for screening tests
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A01K 67/0276 Knock-out vertebrates
• CPC Class Codes: C07K 14/70571 for neuromediators, e.g. serotonin receptor, dopamine receptor
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2830/008 cell type or tissue specific enhancer/promoter combination

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.

Claims

1-39. -39. (Cancelled).

40. A method of screening for a compound that modulates activity of a promoter sequence of the β

-2 subunit of a neuronal nicotinic acetylcholine receptor, comprising;

(a) contacting the compound with a cell line, wherein the cell line is a muscle cell line or a neuronal cell line and comprises in its genome a promoter sequence of the β

-2 subunit of the neuronal nicotinic acetylcholine receptor operatively linked to a heterologous sequence encoding a polypeptide; and

(b) selecting the compound that modulates the activity of the promoter sequence of the β

-2 subunit of the neuronal nicotinic acetylcholine receptor.

41. The method according to claim 40, wherein the heterologous sequence is a reporter gene.

42. The method according to claim 41, wherein the reporter gene encodes β

-galactosidase or luciferase.

43. A method of screening for a compound that increases or decreases activity of a promoter sequence of the β

-2 subunit of a neuronal nicotinic acetylcholine receptor, comprising;

(a) contacting the compound with a cell line, wherein the cell line is a muscle cell line or a neuronal cell line and comprises a promoter sequence of the β

-2 subunit of the neuronal nicotinic acetylcholine receptor operatively linked to a heterologous sequence encoding a polypeptide;

(b) measuring directly or indirectly the expression of the polypeptide; and

(c) selecting the compound that increases or decreases expression of the polypeptide, wherein an increase or decrease in polypeptide expression correlates with an increase or decrease in activity of the promoter sequence of the β

-2 subunit of the neuronal nicotinic acetylcholine receptor.

44. The method according to claim 43, wherein the promoter sequence is selected from the group consisting of:

(A) the nucleic acid sequence from about nucleotide −

1125 to about nucleotide +38 as set forth in FIG. 1 (SEQ ID NO.

     22); and

(B) a sequence having promoter activity, which hybridizes to DNA complementary to the sequence (A) under stringent conditions.

45. The method according to claim 43, wherein the heterologous sequence is a reporter gene.

46. The method according to claim 44, wherein the reporter gene encodes β

-galactosidase or luciferase.

50. The method according to claim 44, wherein the cell line is a neuronal cell line.

47. A method of screening for a compound that increases or decreases activity of a promoter sequence of the β

-2 subunit of a neuronal nicotinic acetylcholine receptor, comprising;

(a) contacting the compound with a cell line, wherein the cell line is a muscle cell line or a neuronal cell line and comprises a promoter sequence of the β

-2 subunit of the neuronal nicotinic acetylcholine receptor, and wherein the promoter sequence is obtained by the process comprising (i) hybridizing a fragment of a β

2-subunit of the neuronal nicotinic acetylcholine receptor gene from a first species with the genomic DNA of a second species under stringent conditions, and (ii) isolating the promoter sequence of the second species from the hybridized sequences;

wherein the promoter sequence of the β

-2 subunit of the neuronal nicotinic acetylcholine receptor is operatively linked to a heterologous sequence encoding a polypeptide;

(b) measuring directly or indirectly the expression of the polypeptide; and

(c) selecting the compound that increases or decreases expression of the polypeptide, wherein an increase or decrease in polypeptide expression correlates with an increase or decrease in activity of the promoter sequence of the β

-2 subunit of the neuronal nicotinic acetylcholine receptor.

48. The method according to claim 47, wherein the heterologous sequence is a reporter gene.

49. The method according to claim 48, wherein the reporter gene encodes β

-galactosidase or luciferase.

51. The method according to claim 48, wherein the cell line is a neuronal cell line.