Efficient construction of gene targeting using phage-plasmid recombination

• US 6,221,647 B1
• Filed: 03/04/1999
• Issued: 04/24/2001
• Est. Priority Date: 10/31/1997
• Status: Expired due to Term

• Current Assignee: Amgen Inc.
• Original Assignee: Amgen Inc.
• Inventors: Woychik, Richard
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US09/262,445
• Time in Patent Office: 782 Days
• Field of Search: 435/456, 435/463, 435/320.1, 435/235.1
• US Class Current: 435/235.1
• CPC Class Codes: C12N 15/907 in mammalian cells

Abstract

A method for producing gene targeting constructs in bacterial by way of homologous recombination between bacterial phage and plasmids.

Claims

1. A method for producing gene targeting constructs in bacteria by single homologous recombination, the method comprising the steps of:

a) preparing a probe plasmid comprising a marker cassette, the marker cassette comprising a suppressor t-RNA gene and a mammalian cell selectable marker, wherein the marker cassette is flanked on at least one side by probe DNA, wherein the probe DNA comprises at least a portion of an exon of a gene to be targeted;

b) introducing the probe plasmid of step a) into a population of homologous recombination proficient suppressor-free bacterial host cells;

c) preparing a target phage, the target phage comprising at least one suppressible mutation in a gene necessary for phage growth and a target DNA comprising a portion of a genomic region to be targeted and wherein the target DNA is homologous to all or part of the probe DNA of step a);

d) infecting the population of bacterial cells of step b) with the phage of step c), allowing recombination between the probe DNA and the target DNA;

e) allowing homologous recombination between the probe plasmid and the target phage; and

f) isolating phage produced in step d).

2. The method of claim 1 step a) wherein the probe sequence comprises from about 15 to 20 kb of genomic DNA.

3. The method of claim 1 wherein the probe DNA flanking the marker cassette comprises from at least about 20 bp to about 40 bp.

4. The method of claim 1 wherein the target DNA comprises from about 15 kb to about 20 kb.

5. The method of claim 1 wherein the suppressor tRNA gene is SupF.

6. The method of claim 1 wherein the mammalian cell selectable marker is a neo gene.

7. The method of claim 6 wherein the neo gene is operatively linked to a promoter which allows expression of the neo gene in embryonic stem cells.

8. The method of claim 1 wherein the phage is a lambda phage.

9. The method of claim 1 wherein the suppressible mutation in the phage is an amber mutation.

10. A targeting construct obtainable by the method of claim 1.

11. A targeting construct produced by the methods of claim 1.

12. A method for obtaining a targeting construct, the method comprising culturing under suitable nutrient and environmental conditions, a population of homologous recombination proficient bacterial cells comprising target phage and a probe plasmid and isolating therefrom, phage resulting from homologous recombination between the target phage and probe plasmid.

13. The method of claim 12 wherein the probe plasmid is a plasmid/SEF.