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.
Claims20
A method of obtaining genetic suppressor elements comprising the steps of:
- (a) randomly fragmenting DNA homologous to a gene to be suppressed, to yield a multiplicity of DNA fragments wherein each DNA fragment comprises an undetermined nucleotide sequence at each end of the fragment and wherein the multiplicity of DNA fragments are produced is representative of the sequence complexity of the gene;
- (b) transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein the expression vector expresses 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 element 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 method of obtaining genetic suppressor elements of a gene that regulates expression of a target gene, the method comprising the steps of:
- (a) randomly fragmenting DNA homologous to the gene to be suppressed, to yield a multiplicity of DNA fragments wherein each DNA fragment comprises an undetermined nucleotide sequence at each end of the fragment and wherein the multiplicity of DNA fragments are produced that is representative of the sequence complexity of the target gene;
- (b) transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein the expression vector expresses the DNA fragments in a living cell in which suppression of the gene that regulates expression of the target gene can be selected or screened;
- (c) genetically modifying living cells by introducing the genetic suppressor element library into the living cells;
- (d) isolating or enriching for genetically modified living cells containing genetic suppressor elements corresponding to the gene that regulates expression of the target gene by selecting or screening for an alteration in expression of the target gene;
- and
- (e) obtaining a genetic suppressor element corresponding to the gene that regulates expression of the target gene from the genetically modified cells.
A method of obtaining living cells containing genetic suppressor elements, comprising the steps of:
- (a) genetically modifying the living cells by introducing a library comprising randomly fragmented DNA sequences, wherein the DNA sequences are homologous to a portion of the gene to be suppressed and comprise a multiplicity of DNA fragments, wherein each DNA fragment comprises an undetermined sequence at each end of the fragment and wherein the multiplicity of DNA fragments are representative of the sequence complexity of the gene, and wherein the library expresses the DNA sequences in the living cell;
- and
- (b) isolating or enriching for genetically modified living cells containing genetic suppressor elements by selecting or screening for gene suppression .
Dependencies are inferred from the claim text.