High throughput gene inactivation with large scale gene targeting

• US 6,069,010 A
• Filed: 09/09/1996
• Issued: 05/30/2000
• Est. Priority Date: 09/11/1995
• Status: Expired due to Fees

• Current Assignee: Axys Pharmaceuticals, Inc. (Quest Diagnostics Incorporated)
• Original Assignee: Axys Pharmaceuticals, Inc. (Quest Diagnostics Incorporated)
• Inventors: Choi, Theodore Kyu
• Primary Examiner(s): Chambers, Jasemine C.
• Assistant Examiner(s): Martin, Jill D.
• Application Number:
  US08/711,218
• Time in Patent Office: 1,359 Days
• Field of Search: 514/44, 800/2, 800/18, 800/21, 800/22, 800/25, 435/252.3, 435/254.2, 435/243, 435/325, 435/172.3, 435/477, 435/471, 435/354, 435/252.33, 435/6
• US Class Current: 435/477
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli

Abstract

The present invention provides a vector system that is useful for the generation of mutations in a recombination-based construction method. The invention further includes the incorporation of mutations generated by the method of the present invention into mouse embryonic stem cells and transgenic mice.

Claims

1. A recombination based method for the generation of a specific disruption in a cloned mammalian genomic sequence of interest, the method comprising:

contacting in a bacterial cell,

(A) a first vector, containing (i) a mammalian genomic DNA sequence of interest of at least about 25 kb in length, (ii) a cleavage site for an endonuclease that cuts once in the vector backbone, (iii) a positive selection marker for said bacterial cell, and (iv) a single copy number origin of replication functional in said bacterial cell, and

(B) a second vector containing (I) a recombination sequence of from about 20 to 1000 bp and having sequence identity with a portion of said genomic DNA sequence of interest, (ii) a positive selection marker for mammalian cells, (iii) a positive selection marker for said bacterial cell, and (iv) a single copy number origin of replication functional in said bacterial cell, that is disparate and compatible with said origin of replication in said first vector and

maintaining said bacterial cell under conditions that promote homologous recombination;

wherein said second vector integrates into said first vector by homologous recombination between said mammalian genomic DNA sequence and said recombination sequence to provide a single recombinant vector comprising an insertion in said mammalian genomic sequence of interest, which recombinant vector is stable and maintained as a single copy in said bacterial cell.

2. A method according to claim 1, wherein said second vector further comprises a bacterial promoter operably linked to a negative selection marker for bacterial cells, and said recombination sequence in said second vector is inserted between said bacterial promoter and said negative selection marker for bacterial cells.

3. A method according to claim 1, wherein said first vector further comprises a negative selection marker for mammalian cells.

4. A method according to claim 1, wherein said bacterial cell further comprises a third vector encoding RecA.

5. A method according to claim 1, wherein said bacterial cell is an E. coli cell.

6. A method for disrupting a mammalian genomic sequence of interest, the method comprising:

contacting in a bacterial cell, (A) a first vector, containing (I) a mouse genomic DNA sequence of interest of at least about 25 kb in length, (ii) a cleavage site for an endonuclease that cuts once in the vector backbone, (iii) a positive selection marker for said bacterial cell, and (iv) a single copy number origin of replication functional in said bacterial cell, and (B) a second vector containing (I) a recombination sequence of from about 20 to 1000 bp and having sequence identity with a portion of said genomic DNA sequence of interest, (ii) a positive selection marker for mammalian cells, (iii) a positive selection marker for said bacterial cell, and (iv) a single copy number origin of replication functional in said bacterial cell, that is disparate and compatible with said origin of replication in said first vector; and

maintaining said bacterial cell under conditions that promote homologous recombination;

wherein said second vector integrates into said first vector by homologous recombination between said mammalian genomic DNA sequence and said recombination sequence to provide a single recombinant vector comprising an insertion in said mammalian genomic sequence of interest, which recombinant vector is stable and maintained as a single copy in said bacterial cell;

isolating said recombinant vector;

linearizing said recombinant vector by cleavage of said cleavage site; and

transfecting said linearized vector into mouse embryonic stem cells comprising said mammalian genomic sequence of interest.

7. A method according to claim 6, further comprising the steps of:

hybridizing said transfected mouse embryonic stem cells with a fluorescently labeled probe prepared from said genomic sequence of interest;

wherein hybridization of said probe in two regions of said transfected mouse embryonic stem cells is indicative of homologous recombination.

8. A bacterial cell comprising:

(A) a first vector, containing (i) a mammalian genomic DNA sequence of interest of at least about 25 kb in length, (ii) a cleavage site for an endonuclease that cuts once in the vector backbone, (iii) a positive selection marker for said bacterial cell, and (iv) a single copy number origin of replication functional in said bacterial cell, and

(B) a second vector containing (i) a recombination sequence of from about 20 to 1000 bp and having sequence identity with a portion of said genomic DNA sequence of interest, (ii) a positive selection marker for mammalian cells, (iii) a positive selection marker for said bacterial cell, and (iv) a single copy number origin of replication functional in said bacterial cell, that is disparate and compatible with said origin of replication in said first vector.

9. A bacterial cell according to claim 8, wherein second vector further comprises a bacterial promoter operably linked to a negative selection marker for bacterial cells and said recombination sequence in said second vector is inserted between said bacterial promoter and said negative selection marker for bacterial cells.

10. A bacterial cell according to claim 9, wherein said negative selection marker is SacB.

11. A bacterial cell according to claim 8, wherein said first vector further comprises a negative selection marker for mammalian cells.

12. A bacterial cell according to claim 8, wherein said bacterial cell further comprises a third vector encoding RecA.

13. A bacterial cell according to claim 8, wherein said bacterial cell is an E. coli cell.

14. A bacterial cell according to claim 8, where said first vector and said second vector single copy origins of replication functional in said bacterial cell are P1 origin and F-factor origin.