Method for identifying cellular targets

A method for identifying a cDNA encoding a product that interacts with components of a signaling pathway that controls the activity of a regulatory module comprising

• introducing an expression system comprising a first nucleic acid construct into a cell, wherein said construct comprises (i) a first nucleic acid molecule comprising a regulatory module operably linked to a first basal promoter operably linked to a sequence encoding a first selectable marker and (ii) a second nucleic acid molecule comprising a basal promoter operably linked to a sequence encoding a second selectable marker, and wherein said first and second nucleic acid molecules are both operably linked to an insulator sequence such that said regulatory module does not affect expression controlled by said second basal promoter;
• introducing a second nucleic acid construct into said cell wherein said construct comprises a cDNA of an expression library wherein said cDNA is operably linked to regulatory elements capable of directing its expression;
• allowing expression of said cDNA;
• contacting said cell with an agent that selects for expression of said first selectable marker and an agent that selects for expression of said second selectable marker;
• and
• determining the viability of said cell wherein a viable cell is identified as containing a cDNA encoding a product that interacts with components of a signaling pathway controlling the activity of said regulatory module.

A method for identifying a cDNA encoding a product that interacts with components of a signaling pathway that is modulated by a chemical compound comprising

• introducing an expression system comprising a first nucleic acid construct into a cell, wherein said construct comprises (i) a first nucleic acid molecule comprising a regulatory module operably linked to a first basal promoter operably linked to a sequence encoding a first selectable marker and (ii) a second nucleic acid molecule comprising a basal promoter operably linked to a sequence encoding a second selectable marker, and wherein said first and second nucleic acid molecules are both operably linked to an insulator sequence such that said regulatory module does not affect expression controlled by said second basal promoter;
• introducing a second nucleic acid construct into said cell wherein said construct comprises a cDNA of an expression library wherein said cDNA is operably linked to regulatory elements capable of directing its expression;
• allowing expression of said cDNA;
• contacting said cell with said compound to modulate said signaling pathway, an agent that selects for expression of said first selectable marker, and an agent that selects for expression of said second selectable marker;
• and
• determining the viability of said cell wherein a viable cell is identified as containing a cDNA encoding a product that interacts with components of a signaling pathway modulated by said chemical compound.

A method for identifying a cDNA encoding a product that interacts with components of a signaling pathway that controls the activity of a regulatory module comprising

• introducing an expression system comprising a first nucleic acid construct into a cell, wherein said construct comprises a nucleic acid molecule comprising a regulatory module operably linked to a basal promoter operably linked to a sequence encoding a fluorescently detectable reporter;
• introducing a second nucleic acid construct into said cell wherein said construct comprises a cDNA of an expression library wherein said cDNA is operably linked to regulatory elements capable of directing its expression;
• allowing expression of said cDNA;
• contacting said cell with an agent that selects for expression of said first detectable reporter;
• and
• identifying a cell as containing a cDNA encoding a product that interacts with components of a signaling pathway controlling the activity of said regulatory module by detecting fluorescence of said reporter in said cells.