Methods and compositions for preparing a genomic library for knockout targeting vectors

• US 6,503,712 B1
• Filed: 05/10/2000
• Issued: 01/07/2003
• Est. Priority Date: 05/10/2000
• Status: Expired due to Fees

• Current Assignee: Amgen Inc.
• Original Assignee: Amgen Inc.
• Inventors: Thukral, Sushil K.
• Primary Examiner(s): McKane; Joseph K.
• Assistant Examiner(s): FRIEND, TOMAS H F
• Application Number:
  US09/569,975
• Time in Patent Office: 972 Days
• Field of Search: 536/23.1 435/6,471,481,483,488,DIG. 23,DIG. 47 935/80
• US Class Current: 506/14
• 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 2267/03 Animal model, e.g. for test or diseases
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic

Abstract

The present invention is directed to methods for producing gene targeting constructs by homologous recombination using mouse genomic libraries arrayed in yeast shuttle vectors. The invention is also directed to methods of using targeting constructs made by the methods to generate transgenic animals.

Claims

1. A method of preparing a genomic library for use in producing knockout targeting vectors comprising:

a) preparing genomic DNA fragments of pre-selected sizes wherein said genomic DNA comprises mouse genomic DNA fragments ranging from 8 kb to 14 kb;

b) preparing a shuttle vector comprising inserting said genomic DNA fragments into a yeast vector wherein said yeast vector is pYYL-1, which comprises;

i) a bacterial origin of replication;

ii) a bacterial selection marker;

iii) a yeast origin of replication;

iv) a yeast selection marker; and

v) a selectable marker for selection of mammalian cells;

c) introducing said vector into bacterial host cells to amplify said shuttle vectors in transformed bacterial host cells; and

;

d) arraying said transformed bacterial host cells into pools wherein the bacterial host cells comprise said shuttle vectors and wherein said pools comprise genomic fragments of pre-selected sizes

wherein said pYYL-1 vector comprises a 4.4 kb fragment, generated through a SphI restriction digestion of yeast-E. coli shuttle vector pGBT9, wherein said 4.4 kb fragment is annealed at the SphI sites to a first oligonucleotide containing a SP6 site and a second oligonucleotide containing a T7 site.

2. The method of claim 1, wherein said mouse genomic DNA fragments are isolated from a mouse strain selected from the group consisting of 129svj, 129 Ola, 129sv, and C57BL/6.

3. The method of claim 1, wherein said genomic fragments of step (a) generate between 3×

10⁶ and 5×

10⁶ clones.

4. The method of claim 1, wherein said host cells are bacterial cells selected from the group consisting of Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Salmonella typhiniurium and Serratia marcescans.

5. The method of claim 4, wherein said host cell is E. coli.

6. The method of claim 1, wherein said bacterial origin of replication is selected from the group consisting of ColE1-ORI, F and R1 bacterial origin of replication.

7. The method of claim 1, wherein said bacterial origin of replication is an E. coli origin of replication.

8. The method of claim 7, wherein said E. coli origin of replication is ColE1-ORI.

9. The method of claim 1, wherein said yeast origin of replication is selected from the group consisting of Cen, 2μ

and the autonomous replication sequence.

10. The method of claim 1, wherein said marker for bacterial propagation is selected from the group consisting of ampicillin resistance, tetracycline resistance, neomycin resistance, kanamycin resistance and chloramphenicol resistance.

11. The method of claim 10, wherein said marker for ampicillin resistance is BlaI.

12. The method of claim 1, wherein said marker for propagation in yeast is selected from the group consisting of trp1, His, Ura3, Arg, Ade and Leu2.

13. The method of claim 1, wherein said selectable marker for mammalian cells is selected from the group consisting of neomycin resistance, hygromycin resistance, zeocin resistance, Salmonella HisD and puromycin-acetyl transferase.

14. The method of claim 1, further comprising a negative selectable marker.

15. The method of claim 14, wherein said negative selectable marker is a negative selectable marker for mammalian cells and is selected from the group consisting of thymidine kinase and xanthine-guanine-phosphoribosyltransferase.

16. The method of claim 1, wherein said yeast vector further comprises a BamHI site for inserting said genomic fragments.

17. The method of claim 16, wherein said BamHI site is flanked by SP6 and T7 priming sequences to facilitate PCR amplification.

18. The method of claim 1, wherein said shuttle vector further comprises rare cutting enzyme sites selected from the group consisting of NotI PacI and SalI flanking the genomic fragment.

19. The method of claim 15, wherein said shuttle vector comprises rare cutting restriction enzyme sites selected from the group consisting of NotI PacI and SalI flanking the mammalian selection marker.

20. A genomic library prepared according to the method of claim 1.

21. A method of preparing a genomic library comprising:

a) preparing size selected genomic DNA ranging from 8 kb to 14 kb in size;

b) preparing a shuttle vector comprising said genomic DNA and a the pYYL-1 yeast vector, wherein said pYYL-1 vector comprises a 4.4. kb fragment, generated through a SphI restriction digestion of yeast-E. coli shuttle vector pGBT9, wherein said 4.4 kb fragment is annealed at the SphI sites to a first oligonucleotide containing a SP6 site and a second oligonucleotide containing a T7 site;

c) amplifying said shuttle vector in transformed bacterial host cells; and

d) arraying said transformed bacterial host cells into pools wherein the bacterial host cells comprise said shuttle vectors and wherein said pools comprise a library of genomic fragments of pre-selected sizes.

22. A genomic library prepared according to the method of claim 21.

23. The genomic library of claim 22, wherein said library is a mammalian genomic library.

24. The genomic library of claim 23, wherein said mammalian genomic library is a mouse genomic library.