Patent ID: 6645767
Filing Date: 2003-11-11
Classification: C12N,C12P

Abstract:
A method of stably inserting at least one gene of interest into a specific locus in the genome of a host haploid yeast cell without adding any expressed bacterial sequences to said host to form a genetically modified diploid, comprising:constructing host haploid yeast strains of opposite mating types to contain a non-lethal, selectable gene mutation, a target gene mutation, in at least one gene of either of said haploids; constructing at least one or a suite of a plurality of yeast integration plasmids, wherein each member plasmid comprises, a selection gene marker which allows for replication of said plasmid in a bacterial plasmid amplification host and for selection of the transformed colonies of said host; a targeting gene marker for directing the insertion of said plasmid into a specific mutated homologous locus, a target gene mutation, in said host yeast genome; a gene of interest that is desired to be expressed in said host yeast; and a bacterial origin of DNA replication functional in said bacterial plasmid amplification host, such that said yeast integration plasmids contain neither any expressed bacterial sequence nor any yeast origin of replication; amplifying each of said yeast integration plasmids in said plasmid amplification host wherein said plasmid amplification host has a selectable phenotype which is complemented by the selection gene marker of said yeast integration plasmid upon its transformation into said plasmid amplification host; purifying said amplified yeast integration plasmids; transforming opposite mating types of said host yeast haploids with yeast integration plasmids such that a targeting gene marker of a yeast integration plasmid directs the integration of said integration plasmid to said target gene mutation in said host to which it is homologous; and mating the transformed haploid yeast cells of opposite mating types to form a diploid cell.