Patent ID: 6204062
Filing Date: 2001-03-20
Classification: C12N

Abstract:
A method of precisely replacing a target sequence in a chromosomal gene sequence of a eukaryotic cell, comprising:providing a linear DNA molecule having (i) a first sequence which is a desired replacement sequence, (ii) second and third sequences substantially homologous to non-identical portions of the chromosomal gene and having proximal and distal ends, the proximal ends flanking the desired replacement sequence, and (iii) a fourth sequence that confers a lethal phenotype and is located at a site selected from the group consisting of the distal end of the second sequence and the distal end of the third sequence;adding a terminating nucleotide analog to each end of the DNA molecule;presenting the resulting DNA molecule to the eukaryotic cell such that the resulting DNA molecule enters and transforms the cell, and replaces the target sequence in the chromosomal gene by a precise double cross-over consisting of a recombination event between each of the second and third sequences of the vector with the homologous portions of the chrornosomal gene, so as to inactivate a function of the chromosomal target gene; andgrowing the cell to obtain recombinant progeny cells having the. desired replacement sequence inserted into the targeted chromosornal gene by the precise double cross-over recombination event.