Converting diploidy to haploidy for genetic diagnosis
AssigneeThe Johns Hopkins University
InventorsBert Vogelstein, Kenneth W. Kinzler, Nickolas Papadopoulos, Hai Yan
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Abstract
Detection of mutations associated with hereditary diseases is complicated by the diploid nature of mammalian cells. Mutations present in one allele are often masked by the wild-type sequence of the other allele. Individual alleles can be isolated from every chromosome within somatic cell hybrids generated from a single fusion. Nucleic acids from the hybrids can be analyzed for mutations in an unambiguous manner. This approach was used to detect two cancer-causing mutations that had previously defied genetic diagnosis. One of the families studied, Warthin Family G, was the first kindred with a hereditary colon cancer syndrome described in the biomedical literature.
Claims42
A method of detecting mutations in a gene of interest of a nonrodent mammal, comprising the steps of:
- fusing cells of a non-rodent mammal to rodent cell recipients of a chromosomally stable cell line to form non-rodent mammal-rodent cell hybrids;
- selecting for fused cell hybrids by selecting for a first marker contained on a rodent chromosome and for a second marker contained on a first non-rodent mammalian chromosome, to form a population of fused cell hybrids;
- detecting among the population of fused cell hybrids a subset of hybrids which are haploid for a second non-rodent mammalian chromosome which is not the same chromosome as the first non-rodent mammalian chromosome and which was not selected;
- analyzing said subset of hybrids to detect a mutation in an mRNA or protein product of a gene or in said gene, wherein the gene resides on the second non-rodent mammalian chromosome.
A method of providing test cells for genetic testing, wherein said test cells are haploid for genes of a non-rodent mammal, comprising the steps of:
- fusing cells of a non-rodent mammal to transformed, diploid, rodent cell recipients of a chromosomally stable cell line to form non-rodent mammal-rodent cell hybrids;
- selecting for fused cell hybrids by selecting for a marker on each of a first nonrodent mammalian chromosome and a rodent chromosome, whereby a population of cells are formed which stably maintain one or more non-rodent mammalian chromosomes in the absence of selection for the non-rodent mammalian chromosomes;
- detecting among the population of cells those cells which are haploid for a second and third non-rodent mammalian chromosome which are distinct from the first non-rodent mammalian chromosome and which were not selected.
Dependencies are inferred from the claim text.