Efficient construction of gene targeting using phage-plasmid recombination

• US 6,090,629 A
• Filed: 10/31/1997
• Issued: 07/18/2000
• 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:
  US08/962,313
• Time in Patent Office: 991 Days
• Field of Search: 435/472, 435/477, 435/235.1
• US Class Current: 435/472
• 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 targeting constructs by double homologous recombination, the method comprising the steps of:

a) preparing a circular probe plasmid comprising a marker cassette and said marker cassette comprising a suppressor t-RNA gene, a mammalian cell selectable marker, said marker cassette flanked on each side by probe DNA homologous to a gene to be targeted and a linker DNA, said linker DNA linking the probe DNA flanking said marker cassette so as to form a circular plasmid;

b) introducing the probe plasmid of step 1 into a population of recombination proficient suppressor-free bacterial host cells;

c) preparing a target phage, said 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 said target sequence 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 bacteriophage of step c), thereby allowing recombination between the probe DNA and the target DNA;

e) isolating phage produced in step d).

2. The method of claim 1 wherein said probe DNA comprises from about at least 40 to about 100 nucleotides.

3. The method of claim 1 wherein said probe DNA each comprise from about at least 20 to about 40 nucleotides.

4. The method of claim 1 wherein said suppressor t-RNA gene is SupF.

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

6. The method of claim 4 wherein said neo gene is operatively linked to a promoter capable of directing expression of the neo gene in embryonic stem cells.

7. The method of claim 5 wherein the promoter is the phosphoglycerate kinase promoter.

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

9. The method of claim 1 wherein the suppressible mutations are amber mutations.