Olfactory receptor expression libraries and methods of making and using them

• US 6,492,143 B1
• Filed: 12/17/1999
• Issued: 12/10/2002
• Est. Priority Date: 12/17/1998
• Status: Expired due to Term

• Current Assignee: Johns Hopkins University
• Original Assignee: Johns Hopkins University
• Inventors: Krautwurst, Dietmar, Yau, King-Wai, Reed, Randall R.
• Primary Examiner(s): Martinell, James
• Application Number:
  US09/465,901
• Time in Patent Office: 1,089 Days
• Field of Search: 536/24.3, 435/91.2, 435/91.21
• US Class Current: 435/91.2
• 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 2227/10 Mammal
• CPC Class Codes: A01K 2267/0393 Animal model comprising a reporter system for screening tests
• CPC Class Codes: B01J 2219/00707 separated from the reactor apparatus
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/32 fusions with soluble part of a cell surface receptor, "decoy receptors"
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: G01N 2500/20 cell-free systems
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5014 for testing toxicity
• CPC Class Codes: G01N 33/5058 Neurological cells
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• CPC Class Codes: G01N 33/6872 Intracellular protein regulatory factors and their receptors, e.g. including ion channels

Abstract

This invention provides novel libraries of olfactory receptor odorant/ligand-binding domains and methods of making and using them. The invention also provides libraries of vectors and cells comprising these nucleic acid constructs. The compositions and methods of the invention are used to identify novel ligand-binding domains for olfactory neuron odorant receptors and their ligands. Thus, the compositions and methods of the invention can be used to generate novel odorants, to screen for toxic odorants, or to manipulate an animal'"'"'s olfactory response.

Claims

1. An amplification primer sequence pair for amplifying a nucleic acid encoding an olfactory receptor ligand-binding region comprising a first primer comprising a sequence 5′

-GGGGTCCGGAG(A/G)(C/G)(A/G)TA(A/G/T)AT(A/G/P)A(A/G/P)(A/G/P)GG-3′

(SEQ ID NO;

1) and a second primer comprising a sequence 5′

-GGGGCTGCAGACACC(A/C/G/T)ATGTA(C/T)(C/T)T(A/C/G/T)TT(C/T)(C/T)T-3′

(SEQ ID NO;

2).

2. A method for generating nucleic acid sequence that encodes a ligand-binding region of an olfactory receptor, the method comprising amplification of a nucleic acid using a primer pair as set forth in claim 1.

3. The method of claim 2, wherein the amplified nucleic acid is genomic DNA, mRNA or cDNA derived from olfactory neurons or olfactory epithelium.

4. The method of claim 2, wherein the amplification comprises the following conditions and steps in the following order:

about one cycle at about 94°

C. for about 2 min; and

about 30 cycles of about 45°

C. to about 65°

C. for about 1 min, followed by about 72°

C. for about one min. followed by about 94°

C. for about 1 min.

5. The method of claim 4, wherein the PCR amplification further comprises the following conditions and steps in the following order:

about one cycle of about 45°

C. to about 65°

C. for about 10 min; and

about one cycle of about 72°

C. for about 10 min.

6. A kit for amplification of olfactory receptor sequences comprising the primer pair of claim 1.