US 6,783,985 B1 · Gene disruption methodologies for drug target discovery

Abstract

The present invention provides methods and compositions that enable the experimental determination as to whether any gene in the genome of a diploid pathogenic organism is essential, and whether it is required for virulence or pathogenicity. The methods involve the construction of genetic mutants in which one allele of a specific gene is inactivated while the other allele of the gene is placed under conditional expression. The identification of essential genes and those genes critical to the development of virulent infections, provides a basis for the development of screens for new drugs against such pathogenic organisms. The present invention further provides Candida albicans genes that are demonstrated to be essential and are potential targets for drug screening. The nucleotide sequence of the target genes can be used for various drug discovery purposes, such as expression of the recombinant protein, hybridization assay and construction of nucleic acid arrays. The uses of proteins encoded by the essential genes, and genetically engineered cells comprising modified alleles of essential genes in various screening methods are also encompassed by the invention.

Claims29

Dependent claims

A method for constructing a strain of diploid fungal cells in which both alleles of a gene are modified, the method comprising the steps of:

(a) modifying a first allele of a gene in diploid fungal cells by recombination using a gene disruption cassette comprising a first nucleotide sequence encoding a first expressible selectable marker, thereby providing heterozygous diploid fungal cells in which the first allele of the gene is inactivated;
and
(b) modifying the second allele of the gene in the diploid fungal cells by recombination using a promoter replacement fragment comprising a second nucleotide sequence encoding a second expressible selectable marker and a heterologous promoter, such that expression of the second allele of the gene is regulated by the heterologous promoter
wherein at least one of the first and the second expressible selectable marker is a drug-resistance marker,
thereby constructing a strain of diploid fungal cells in which both alleles of a gene are modified.

2 more elements

A method of assembling a collection of diploid fungal cells each of which comprises modified alleles of a different gene, the method comprising the steps of:

(a) modifying a first allele of a first gene in diploid fungal cells by recombination using a gene disruption cassette comprising a first nucleotide sequence encoding a first expressible selectable marker, thereby providing heterozygous diploid fungal cells in which the first allele of the gene is inactivated;
(b) modifying a second allele of the first gene in the heterozygous diploid fungal cells by recombination using a promoter replacement fragment comprising a second nucleotide sequence encoding a second expressible selectable maker and a heterologous promoter, such that expression of the second allele of the gene is regulated by the heterologous promoter, wherein at least one of the first and the second expressible selectable marker is a drug-resistance selectable marker, thereby providing a first strain of diploid fungal cells comprising a modified allelic pair of the first gene;
and
(c) repeating steps (a) and (b) a plurality of times, wherein a different gene is modified with each repetition, thereby providing the collection of diploid fungal cells each comprising the modified alleles of a different gene.

1 more element

A strain of diploid fungal cells comprising modified alleles of a gene, wherein the first allele of the gene is inactivated by recombination using a gene disruption cassette comprising a nucleotide sequence encoding a first expressible selectable marker the second allele of the gene is modified by recombination using a promoter replacement fragment comprising a second nucleotide sequence encoding a second expressible selectable marker and a heterologous promoter such that expression of the second allele of the gene is regulated by the heterologous promoter that is operably linked to the coding region of the second allele of the gene;

wherein the first modified allele of the gene in the strain is linked with the first expressible selectable marker and the second modified allele of the gene in the strain is linked with the second expressible selectable marker;
and wherein at least one expressible selectable marker in the strain is a drug-resistance selectable marker.

Dependencies are inferred from the claim text.

Gene disruption methodologies for drug target discovery

Life of this patent

Gene disruption methodologies for drug target discovery

Life of this patent

Abstract

Claims29