METHOD FOR INSERTING GENETIC MATERIAL INTO GENOMIC DNA

摘要

The present invention provides reagents and methods for improved homologous recombination.

主权项

1. A method for homologous recombination, comprising
(a) expressing in a host cell a first nucleic acid encoding a coumermycin dimerization domain of E. coli gyrase B fused to a catalytic domain of mammalian dsRNA dependent protein kinase, PKR (GyrB-PKR), wherein the first nucleic acid is operatively linked to nucleic acid control sequences, and wherein the first nucleic acid is present on a first nucleic acid construct that further comprises:
(i) a first homologous recombination site flanking the first nucleic acid; and(ii) a second homologous recombination site flanking the first nucleic acid; wherein the first homologous recombination site and the second homologous recombination site are not capable of recombining with each other; (b) expressing in the host cell a second expression vector comprising a gene of interest flanked by the first homologous recombination site and the second homologous recombination site, wherein the gene of interest is operatively linked to nucleic acid control sequences; and (c) culturing the cells in medium comprising coumermycin under conditions suitable to cause host cell death if homologous recombination between the first expression vector and the second expression vector has not occurred.