METHOD FOR GENE ISOLATION BY CRE-TRAP CLONING

摘要

The disclosure describes a method for isolating genes encoding proteins thatregulate the expression of target genes of interest, said method designatedherein for convenience by the term: "Cre-Trap cloning." In brief, the Cre-trapcloning method of the invention involves modifying a target gene of interestsuch that it encodes the Cre recombinase and Herpes simplex virus (HSV)thymidine kinase (TK) proteins. This step is followed by mutagenesis andselection for cells which have lost target gene expression by virtue of theirresistance to ganciclovir (a nucleoside analog structurally related toacyclovir), which kills cells expressing HSV TK. Cell lines that have losttarget gene expression due to mutations in genes encoding traps-acting factorsare then transiently transfected with a cDNA library from the parent cell linein a novel expression vector (pCT.l, shown in Figure 1). Expression of a cDNAthat complements the genetic mutation results in expression of the target geneand production of the Cre recombinase, which modifies pCT.I in such a way thatit is readily isolated from pCT.I plasmids with cDNAs that do not activatetarget gene expression.