Genes and genetic elements associated with sensitivity to chemotherapeutic drugs
AssigneeBoard of Trustees of the University of Illinois-Urbana
InventorsIgor B. Roninson, Andrei Gudkov
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Abstract
The invention provides genetic suppressor elements that confer upon a cell resistance to one or more chemotherapeutic drug, methods for identifying and obtaining such elements, and methods of using such elements. The invention also provides cloned genes associated with sensitivity to chemotherapeutic drugs.
Claims93
A method of identifying genetic suppressor elements (GSEs) that confer a selectable phenotype upon a eukaryotic cell, the method comprising the steps of:
- (a) synthesizing randomly fragmented cDNA from the total mRNA of a eukaryotic cell to yield DNA fragments having a size of less than about 700 basepairs;
- (b) transferring the DNA fragments to an expression vector to yield a library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector which expresses the DNA fragments in a living eukaryotic cell that which exhibits the selectable phenotype;
- (c) genetically modifying living eukaryotic cells by introducing the genetic suppressor element library into the cells;
- (d) isolating or enriching for genetically modified living eukaryotic cells containing genetic suppressor elements which centers the selectable phenotype by selecting cells that express the selectable phenotype, and;
- (e) obtaining the genetic suppressor elements from the genetically modified cells.
A method of identifying GSEs corresponding to genes that when suppressed by GSEs, confer a selectable phenotype upon a eukaryotic cell having such suppressed genes, the method comprising the steps of:
- (a) obtaining a total mRNA population from a eukaryotic cell;
- (b) synthesizing randomly fragmented cDNA from the total mRNA to yield random cDNA fragments having a size less than about 700 basepairs;
- (c) ligating the random cDNA fragments to synthetic adaptors to produce amplifiable random cDNA fragments;
- (d) amplifying the random cDNA fragments;
- (e) cloning the amplified mixture of random cDNA fragments into a suitable expression vector to produce a random fragment expression library, wherein each of the random cDNA fragments is operatively linked to a protein translation initiation codon;
- (f) transferring the random fragment expression library into appropriate target eukaryotic cells;
- (g) isolating or enriching for genetically modified living cells containing a selectable phenotype-conferring genetic suppressor elements by identifying and isolating or enriching for those cells that express the selectable phenotype;
- and
- (h) recovering the GSE from the target cell having the selectable phenotype.
A method for identifying GSEs corresponding to genes that, when suppressed by GSE, confer a selectable phenotype upon a eukaryotic cell having such suppressed genes, the method comprising the steps of:
- (a) obtaining genomic DNA from a eukaryotic cell;
- (b) randomly fragmenting the genomic DNA to produce random genomic DNA fragments having a size less than about 700 basepairs;
- (c) ligating the random genomic DNA fragments to synthetic adaptors to produce amplifiable random genomic DNA fragments;
- (d) amplifying the random genomic DNA fragments;
- (e) cloning the amplified mixture of random genomic DNA fragments into a suitable expression vector to produce a random fragment expression library, wherein each of the random genomic DNA fragments is operatively linked to a protein translation initiation codon;
- (f) transferring the random fragment expression library into appropriate target eukaryotic cells;
- (g) isolating or enriching for genetically modified living cells containing a selectable phenotype-conferring genetic suppressor element by identifying and isolating or enriching for those cells that express the selectable phenotype;
- and
- (h) recovering the GSE from the target cell having the selectable phenotype.
A method of identifying genetic suppressor elements that confer upon a eukaryotic cell resistance to one or more chemotherapeutic drugs, the method comprising the steps of:
- (a) obtaining random DNA fragments of a gene associated with sensitivity to chemotherapeutic drugs having a size less than about 700 basepairs;
- (b) transferring the DNA fragments to an expression vector to yield a library, wherein each of the random DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector is capable of expressing the DNA fragments in a living eukaryotic cell that is susceptible of inhibitory effects of a chemotherapeutic drug;
- (c) genetically modifying living eukaryotic cells by introducing the genetic suppressor element library into the cells;
- (d) isolating or enriching for genetically modified living cells containing a chemotherapeutic drug resistance-conferring genetic suppressor element by selecting cells in the presence of chemotherapeutic drug, and;
- (e) obtaining a genetic suppressor element from the genetically modified eukaryotic cells.
A normalized library of randomly fragmented cDNA corresponding to total mRNA expressed in a mammalian cell wherein each of the fragments does not exceed about 700 basepairs in length and wherein the random cDNA fragments are carried within a vector capable of expressing the fragments as a mRNA in a mammalian cell, each of the fragments being operatively linked to a translation initiation codon.
Dependencies are inferred from the claim text.