Methods and compositions for elucidating protein expression profiles in cells
OwnerCharles Link, Tatiana Seregina +8inventors
InventorsCharles Link, Tatiana Seregina, Nicholas Vahanian, James Higginbotham, William Ramsey, Bradley Powers, Sachet Shukla, Won Young, Teresa Dicolandrea, Mario Mautino
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Abstract
The present invention relates generally to methods and compositions for the identification of differential protein expression patterns and concomitantly the active genetic regions that are directly or indirectly involved in different tissue types, disease states, or other cellular differences desirable for diagnosis or for targets for drug therapy.
Claims32
A method for elucidating a protein expression profile of a test cell line or group of cells, the method comprising:
- randomly introducing into the genome of a cell or group of cells a promoterless polynucleotide construct, the construct comprising in a 5′ to 3′ orientation:
- i) a splice acceptor consensus sequence;
- ii) a complementary sequence of a first type IIS restriction enzyme recognition sequence;
- iii) an oligonucleotide sequence encoding an assayable marker peptide;
- iv) a sequence of a second type IIS restriction enzyme recognition sequence;
- v) a splice donor consensus sequence;
- wherein said promoterless polynucleotide construct when introduced into an actively expressed gene results in the generation of a fusion protein, containing the assayable marker peptide inserted at a random position within two exons coding for the cellular protein encoded by said gene;
- vi) identifying those cells expressing said marker peptide fused to said cellular protein;
- vii) determining the identity of the proteins to which the marker peptide is fused in each group of cells.
A method to identify differentially expressed proteins in two different populations of cells, the method comprising:
- randomly introducing into the genomes of a reference group of cells and into the genomes of a test group of cells a promoterless polynucleotide construct, wherein the construct comprises, in a 5′ to 3′ orientation;
- i) a splice acceptor consensus sequence;
- ii) a complementary sequence of a first type IIS restriction enzyme recognition sequence;
- iii) an oligonucleotide sequence encoding an assayable marker peptide;
- iv) a sequence of a second type IIS restriction enzyme recognition sequence;
- v) a splice donor consensus sequence;
- thereby generating a population of cells, each bearing a different cellular protein with an assayable peptide marker inserted at random positions within each fusion protein;
- vi) identifying those cells expressing said marker peptide fused to said cellular protein in each group of cells;
- vii) determining the identity of the proteins to which the marker peptide is fused in each group of cells;
- and
- viii) comparing by statistical methods the protein expression profiles obtained for the test group of cells against the protein expression profiles obtained for the reference group of cells, thereby identifying differences in the expression levels of fusion proteins among the two groups of cells.
Dependencies are inferred from the claim text.