High throughput knockout constructs in yeast by homologous recombination

• US 20030104456A1
• Filed: 11/08/2002
• Published: 06/05/2003
• Est. Priority Date: 05/10/2000
• Status: Abandoned Application

• Current Assignee: Sushil K. Thukral
• Original Assignee: Sushil K. Thukral
• Inventors: Thukral, Sushil K.
• Application Number:
  US10/291,022
• Publication Number:
  US 20030104456A1
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 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;

b) preparing a shuttle vector comprising inserting said genomic DNA fragments into a yeast vector, wherein the vector comprises;

i) a first bacterial origin of replication;

ii) a first bacterial selection marker;

iii) a first yeast origin of replication;

iv) a first yeast selection marker; and

v) a first mammalian selection marker;

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.

2. The method of claim 1, wherein said shuttle vectors in said bacterial host cells comprise:

vi) a genomic DNA fragment;

ii) a first yeast origin of replication;

iii) a first yeast selection marker;

iv) a first bacterial origin of replication;

v) a first bacterial selection marker; and

vi) a first selection marker for integration into mammalian cells.

3. The method of claim 1, wherein said genomic DNA comprises mouse genomic DNA fragments ranging from about 8 kb to about 14 kb.

4. The method of claim 3, 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.

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

10⁶ and 5×

10⁶ clones.

6. 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 typhimurium and Serratia marcescans.

7. The method of claim 6, wherein said host cell is E. coli.

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

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

10. The method of claim 9, wherein said E. coli origin of replication is ColE1-ORI.

11. The method of claim 1, wherein said yeast origin of replication is selected from the group consisting of Cen, 2 m and the autonomous replication sequence.

12. 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.

13. The method of claim 12, wherein said marker for ampicillin resistance is BlaI.

14. 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.

15. 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.

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

17. The method of claim 16, wherein said negative selectable marker is selected from the group consisting of thymidine kinase, and xanthine-guanine-phosphoribosyltransferase.

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

19. The method of claim 18, wherein said BamHI site is flanked by priming sequences to facilitate PCR amplification.

20. The method of claim 19, wherein said priming sequences are Sp6 and T7 priming sequences.

21. The method of claim 1, wherein said yeast vector is designated pYYL-1.

22. The method of claim 1, wherein said shuttle vector further comprises rare cutting enzyme sites flanking the genomic fragment.

23. The method of claim 17, wherein said shuttle vector comprises rare cutting enzyme sites flanking the mammalian selection marker.

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

25. The genomic library of claim 24, wherein said genomic library is used for high throughput construction of knockout vectors.

26. The library of claim 24, wherein said genomic library is used for high throughput construction of mouse knockout vectors.

27. A method of preparing a gene targeting vector for homologous recombination comprising:

a) selecting a bacterial clone pool positive for the gene to be targeted from an array of bacterial clones comprising the genomic library said array prepared according to the method of claim 1;

b) isolating the DNA from said positive pool;

c) preparing a second expression construct comprising a marker cassette comprising a second yeast selectable marker and a second mammalian selectable marker, wherein said marker cassette is flanked on each side by mammalian gene-specific sequences homologous for a portion of the gene to be targeted;

d) transforming yeast cells with the second expression construct and the DNA from the positive clone;

e) selecting the transformed yeast cells for expression of the first and second yeast selectable markers; and

f) isolating the targeting vector produced by the recombination between the shuttle vector and the second expression construct.

28. The method of claim 27, wherein said positive pools comprising the target gene are selected by PCR analysis of the pools with gene-specific PCR primers wherein amplification of the PCR products is indicative of the pool comprising the target gene of interest.

29. The method of claim 27, wherein said gene-specific flanking sequences each comprises at least about 20 nucleotides.

30. The method of claim 27, wherein said gene specific flanking sequences each comprises from about 35 to about 400 nucleotides.

31. The method of claim 27, wherein said fragment of genomic DNA comprises from about 0.5 kb to about 5 kb of DNA on each side of a site in said gene to be targeted.

32. The method of claim 27, wherein said fragment of genomic DNA comprises at least about 1 kb of genomic DNA on each side of a site in said gene to be targeted.

33. The method of claim 27, wherein said second yeast selectable marker is selected from the group consisting of TRP1, His, Ura3, Ade, Arg and Leu2.

34. The method of claim 27, wherein said second mammalian selectable marker is selected from the group consisting of consisting of thymidine kinase, neomycin resistance, hygromycin resistance, Salmonella HisD and puromycin-acetyl transferase.

35. The method of claim 27, wherein said marker cassette comprises Ura3 as the second yeast selectable marker and the neomycin resistance gene as the second mammalian selectable marker.

36. A method of preparing a genomic library comprising:

a) preparing size selected genomic DNA;

b) preparing a shuttle vector comprising said genomic DNA and a yeast vector;

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.

37. A genomic library prepared according to the method of claim 36.

38. The genomic library of claim 37, wherein said library is a mammalian genomic library.

39. The genomic library of claim 38, wherein said mammalian genomic library is a mouse genomic library.