Stem Cell Related Patent Number US6221647

Efficient construction of gene targeting using phage-plasmid recombination

Woychik, Richard; Beechwood, OH, USA

This invention describes a system of production of gene targeting constructs in bacteria via homologous recombination between bacterial phage and plasmids. Such a method of construction involves the preparation of a probe plasmid comprising a marker cassette, which in turn comprises a suppressor t-RNA gene and a mammalian cell selectable marker. 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. Further disclosed are means of introducing the probe plasmid into the population of homologous, recombination-proficient, suppressor-free bacterial host cells. Also included are the steps for preparing a target phage, such that the target phage comprises at least one suppressible mutation in a gene necessary for phage growth and a target DNA consisting of a portion of a genomic region to be targeted, such that the target DNA is homologous to all or part of the probe DNA. Methods of infection, recombination and isolation are also provided.

Application Number: US1999000262445

Date Filed: 03/04/99

Dated Published: 04/24/01

Assignee: Amgen, Inc., Thousand Oaks, CA, USA