System for F-box hormone receptor regulated protein expression in mammalian cells

A method for F-box hormone receptor regulated target protein degradation in a mammalian host cell comprising:

• providing a mammalian host cell;
• infecting, into the mammalian host cell, a first lentiviral vector comprising (i) a first silencing nucleic acid molecule comprising a primary first promoter and a first shRNA operably linked to the primary first promoter, wherein the first shRNA silences expression of a first target protein and (ii) a first expression nucleic acid molecule comprising a primary second promoter, a Transport Inhibitor Response 1 (TIR1) receptor operably linked to the primary second promoter, and a nucleic acid molecule encoding a first fusion protein comprising an auxin-induced degradation (AID) degron fused to a first target protein, wherein the nucleic acid molecule encoding the first fusion protein is operably linked to the primary second promoter;
• infecting, into the mammalian host cell, a second lentiviral vector comprising (i) a second silencing nucleic acid molecule comprising a secondary first promoter and a second shRNA operably linked to the secondary first promoter, wherein the second shRNA silences expression of a second target protein and (ii) a second expression nucleic acid molecule comprising a secondary second promoter, a Coronatine Insensitive 1 (COI1 receptor operably linked to the secondary second promoter, and a nucleic acid molecule encoding a second fusion protein comprising a jasmonate ZIM-domain (JAZ) degron fused to a second target protein, wherein the nucleic acid molecule encoding the second fusion protein is operably linked to the secondary second promoter;
• culturing said infected mammalian host cell under conditions that result in (i) silencing expression of the first and second target proteins and (ii) expression of the first and second fusion proteins;
• contacting said infected mammalian host cell with a molecule that binds the TIR1 receptor so that said first fusion protein undergoes degradation;
• and
• contacting said infected mammalian host cell with a molecule that binds the COI1 receptor so that said second fusion protein undergoes degradation.