Patent ID: 6228649
Filing Date: 2001-05-08
Classification: A61K,A61P,C07K,C12N,Y02A

Abstract:
A method of producing a genetic mutant transfected with at least three different nucleic acid constructs, the method comprising:a) transfecting a unicellular diploid organism with first and second nucleic acid constructs, said first construct comprising a first selectable marker flanked by nucleotide sequences complementary to 5' and 3' flanking regions of a first allele of a genetic locus to be replaced, and said second construct comprising a second selectable marker flanked by nucleotide sequences complementary to 5' and 3' flanking regions of a second allele of the genetic locus to be replaced under conditions to promote deletion and replacement of said genetic locus by said DNA sequences;b) selecting for replacement and loss of said genetic locus from the genome of said unicellular diploid organism by detecting expression of both said different marker genes;c) isolating a genetic mutant of said unicellular diploid organism that expresses both said selectable marker genes, said mutant having lost both said alleles of said genetic locus via replacement with both said selectable markers; andd) further transfecting said genetic mutant of said unicellular diploid organism with a third nucleic acid construct defining a DNA sequence which encodes an immunogen.