Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Fused or hybrid cell – per se
Reexamination Certificate
1999-12-15
2002-06-04
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Fused or hybrid cell, per se
Reexamination Certificate
active
06399374
ABSTRACT:
BACKGROUND OF THE INVENTION
The problem with humans, at least from a genetic diagnostic perspective, is that they are diploid. Mutations in one allele, such as those responsible for all dominantly inherited syndromes, are always accompanied by the wild-type sequence of the second allele. Though many powerful techniques for genetic diagnosis have been developed over the past decade, all are compromised by the presence of diploidy in the template. For example, the presence of a wild-type band of the same electrophoretic mobility as a mutant band can complicate interpretation of sequencing ladders, especially when the mutant band is of lower intensity. Deletions of a segment of DNA are even more problematic, as in such cases only the wild-type allele is amplified and analyzed by standard techniques. These issues present difficulties for the diagnosis of monogenic diseases and are even more problematic for multigenic diseases, where causative mutations can occur in any of several different genes. Such multigenism is the rule rather than the exception for common predisposition syndromes, such as those associated with breast and colon cancer, blindness, and hematologic, neurological, and cardiovascular diseases. The sensitivity of genetic diagnostics for these diseases is currently suboptimal, with 30% to 70% of cases refractory to genetic analysis.
There is a need in the art for simply separating and analyzing individual alleles from human cells.
SUMMARY OF THE INVENTION
It is an object of the invention to provide a method for detecting mutations in a gene of interest on a human chromosome.
It is another object of the invention to provide a method for making test cells suitable for sensitive genetic testing.
It is yet another object of the invention to provide a population of fused cell hybrids which are useful for genetic analysis.
These and other objects of the invention are provided by one or more of the embodiments described below. In one embodiment a method of detecting mutations in a gene of interest of a human is provided. Cells of a human are fused to rodent cell recipients to form human-rodent cell hybrids. Fused cell hybrids are selected by selecting for a first marker contained on a rodent chromosome and for a second marker contained on a first human chromosome, forming a population of fused cell hybrids. A subset of hybrids are detected among the population of fused cell hybrids. The hybrids are haploid for a second human chromosome which is not the same chromosome as the first human chromosome and which was not selected. The subset of hybrids are tested to detect a gene, an mRNA product of said gene, or a protein product of said gene. The gene resides on the second human chromosome. Diminished amounts of the mRNA or protein product or altered properties of the gene, mRNA, or protein product indicate the presence of a mutation in the gene in the human.
According to another embodiment, a method is disclosed which provides test cells for genetic testing. The test cells are haploid for human genes. Cells of a human are fused to transformed, diploid, rodent cell recipients to form human-rodent cell hybrids. Fused cell hybrids are selected by selecting for a marker on each of a first human chromosome and a rodent chromosome, forming a population of cells which stably maintain one or more human chromosomes in the absence of selection for the human chromosomes. Cells which are haploid for a second human chromosome which is distinct from the first human chromosome are detected among the population of cells; the second human chromosome was not selected.
Also provided by the present invention is a population of rodent-human hybrid cells wherein each homolog of at least 2 human autosomes is present in haploid form in at least one out of one hundred of the cells.
The present invention thus provides the art with a method which can be used to increase the sensitivity and effectiveness of various diagnostic and analytic methods by providing hybrid cells to analyze which are haploid for one or more genes of interest. The human chromosome content of the hybrid cells is stable and uniform.
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Kinzler Kenneth W.
Papadopoulos Nicholas
Vogelstein Bert
Yan Hai
Banner & Witcoff , Ltd.
Sandals William
The Johns Hopkins University
Yucel Remy
LandOfFree
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