Retrovirus promoter-trap vectors
AssigneeMassachusetts Institute of Technology
InventorsH. Earl Ruley, Harold von Melchner
Life of this patent
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Abstract
Retroviruses are used as genetic tools to isolate transcriptionally active chromosomal regions. The retroviruses have a promoterless protein coding sequence located in U3 or U5. The retroviruses may be used to infect cells under conditions which permit selection for instances when the retrovirus integrates in close proximity to and under the control of a cellular promoter. The promoter and its associated gene then may be identified and isolated. In this manner, the retroviruses function as promoter-traps. Related methods and products including vectors, kits and assays are provided.
Claims17
A method for putting a protein coding sequence under the control of an endogenous promoter comprising:
- introducing into living cells promoterless oligonucleotides coding for a protein, each oligonucleotide joined to a DNA integration sequence, wherein said introducing is carried out by infecting said living cells with a retrovirus having a promoterless oligonucleotide coding for a protein located within a U3 or U5 control region of the retrovirus,
- allowing the promoterless oligonucleotides coding for a protein to integrate into genomic DNA, and
- selecting a cell that has one of the promoterless oligonucleotides coding for a protein integrated under the regulatory control of a promoter endogenous to the cell to result in a functional gene fusion.
A method for identifying an endogenous promoter in cellular genomic DNA comprising:
- introducing into living cells promoterless oligonucleotides coding for a protein, each oligonucleotide flanked by a DNA integration sequence, wherein the promoterless oligonucleotide is introduced by infecting the cells with a retrovirus having the promoterless oligonucleotide located in a control region of the retrovirus,
- selecting from said cells at least one cell having incorporated into its genomic DNA the promoterless oligonucleotide coding for a protein in a manner such that the protein coding sequence both is separated from an endogenous promoter by said DNA integration sequence and is regulated by said endogenous promoter, and
- isolating said endogenous promoter or a portion thereof from said genomic DNA.
A cell containing a retrovirus having a promoterless oligonucleotide coding for a protein located within a U3 or U5 control region of the retrovirus.
Dependencies are inferred from the claim text.