Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1999-07-06
2000-05-02
Brusca, John S.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 29, 4353201, C12Q 168
Patent
active
06057111&
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method of identifying genes, specifically genes that maintain specific cell phenotypes.
2. Description of Related Art
There are methods available to isolate and identify specific genes. However these methods are not efficient and rapid. Applicant has previous disclosed the Technical Knock Out (TKO) selection method which has the advantage of rapid isolation of genes that inhibit proliferation in a specified restrictive environment [Deiss and Kimchi, 1991; Deiss et al, 1995; Kissil et al, 1995; Deiss et al, 1996]. However, this method has the limitation of requiring a phenotype that can be efficiently selected against, such as a cell growth arrest or cell killing phenotype.
Recently Smith et al [1995] and U.S. Pat. No. 5,612,180 have described a method called genetic footprinting to identify genes. The method involves mutagenesis of potentially large numbers of genes followed by a genetic selection of the cells containing the mutated genes. This is followed by retrospective analysis of the effect of individual gene inactivation on the behavior cells containing these inactivations. From this information new genes are determined. This method has significant disadvantages for large scale gene identification. The genetic footprinting method involves mutagenesis by gene insertion and because of this requires a haploid target which imposes a limitation on the method. Second, the method of determining the effect of each gene inactivation on the fitness of the cells containing the mutation involves a PCR amplification of the target gene which requires prior knowledge of the nucleotide sequence of all the target genes that will be studied which limits the gene base which can be searched. It would be useful to have a method which does not require a haploid target and does not require a known sequence.
It would be useful to have a rapid method which can identify genes to be isolated that are essential for the maintenance of specific cell phenotypes where positive selection exists for the phenotypes. These identified genes are excellent targets for the development of pharmacological inhibitors which would also act clinically to inhibit the specific phenotype. In other words it would be useful to have a tool which can effectively identify pharmacological targets for inhibition of deleterious phenotypes.
SUMMARY OF THE INVENTION
According to the present invention, a method for the identification of genes that are essential for the maintenance of specific cell phenotypes is disclosed. The method includes the initial step of identifying a cell type with a phenotype of interest. The method allows the phenotype of interest to be phenotypes relating to growth, phenotypes relating to release of factors and phenotypes relating to other basic cell functions.
Gene inactivation is performed on an aliquot of cells of the cell type of interest. Possible methods of gene inactivation include Genetic Suppressor Element (GSE) inactivation, Random Homozygous Knock-Out (RHKO) inactivation, or Technical Knock Out (TKO) inactivation.
Positive selection is then performed on an aliquot of the cell culture to which gene inactivation has been applied. The positive selection includes manipulations that test the ability of cells to survive under specific culture conditions, ability to express a specific factor, changes in cell structure, or differential gene expression.
Cells which continue to maintain the phenotype following gene inactivation have not had the gene of interest inactivated whereas cells in which genes necessary for maintaining the phenotype have been inactivated have been lost. Utilizing subtraction analysis between treated and untreated aliquots the gene in the cells which has been inactivated that affects the phenotype of interest is identified. The subtraction analysis can include the methods of differential display, representational differential analysis (RDA), suppressive subtraction hybridization (SSH), serial analysis of gene expres
REFERENCES:
Deiss et al. Identification of a novel serine/threonine kinase and a novel 15-kD protein as potential mediatiors of a gamma interferon-induced cell death. Genes & Development vol. 9 pp. 15-30, 1995.
Deiss Louis Paul
Efimova Elena
Einat Paz
Vasquez-Iaslop Nora Cecilia
Yehiely Fruma
Brusca John S.
Quark Biotech, Inc.
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