Methods and applications for efficient genetic suppressor elements
AssigneeThe Board of Trustees of the University of Illinois
InventorsIgor B. Roninson, Tatyana Holzmayer, Choi Kyunghee, Andrei Gudkov
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Abstract
Methods for isolating and identifying genetic elements that are capable of inhibiting gene function are disclosed, as well as genetic elements isolated or identified according to the method of the invention and host cells modified by genetic modification using genetic suppressor elements according to the invention.
Claims21
A genetic suppressor element obtained by a method comprising the steps of:
- (a) randomly fragmenting DNA homologous to a gene to be suppressed, to yield DNA fragments;
- (b) transferring the DNA fragments to an expression vector to yield a library, wherein the expression vector is capable of expressing the DNA fragments in a living cell in which gene suppression can be selected or screened;
- (c) genetically modifying living cells by introducing the genetic suppressor elements library into the living cells;
- (d) isolating or enriching for genetically modified living cells containing genetic suppressor elements by selecting or screening for gene suppression, and;
- (e) obtaining the genetic suppressor element from the genetically modified cells.
A synthetic oligonucleotide having a nucleotide sequence corresponding to the nucleotide sequence of an antisense RNA encoded by a genetic suppressor element produced by a method comprising the steps of:
- (a) obtaining genomic DNA or a total cDNA population from the cells;
- (b) randomly fragmenting the genomic DNA or total cDNA population to produce random DNA fragments;
- (c) ligating the random DNA fragments to synthetic adaptors to produce amplifiable random DNA fragments;
- (d) cloning the amplified mixture of random DNA fragments into a suitable expression vector having a selectable marker to produce a random fragment expression library;
- (e) transferring the random fragment expression library into appropriate the random fragment expression library into appropriate target cells;
- (f) selecting the target cells for the presence of the selectable marker present in the expression vector to obtain target cells having the selectable marker;
- (g) selecting or screening the target cells having the selectable marker for the selectable or screenable phenotype conferred upon the cells by inactivation of a recessive gene by a GSE;
- and
- (h) recovering the GSE from the target cell having the selectable or screenable phenotype.
Dependencies are inferred from the claim text.