Patent ID: 6251676
Filing Date: 2001-06-26
Classification: C12N,C12Q

Abstract:
A method for detecting interaction between a bait and a prey in a mammalian cell two hybrid system, comprising:(a) providing a mammalian cell comprising: a reporter gene encoding a fluorescent polypeptide operably linked to an upstream transcriptional regulatory sequence comprising a DNA binding site for a DNA-binding domain, the reporter gene and transcriptional regulatory sequence being integrated into a chromosome of the mammalian cell;(b) introducing into the cell a DNA molecule comprising a first nucleotide sequence encoding a bait fusion protein, comprising an upstream GAL4 DNA-binding domain and the bait;(c) introducing into the cell a DNA molecule comprising a nucleotide sequence encoding prey fusion protein comprising an upstream transcriptional activation domain and the prey, the transcriptional activation domain being selected from the group consisting of a GAL4 transcriptional activation domain, a VP16 transcriptional activation domain, and a CR2 transcriptional activation domain from MSG1;(d) incubating the cell for 96 hours or less;(e) detecting reporter gene expression, if present, thereby detecting interaction between the bait and the prey.