Team:LMU-Munich/Jump-or-die

Jump-or-Die System

This system also uses Bak as apoptosis inducing selection marker together with the tet-on promoter, which is to be stably integrated in the cell-line genome. However, this construct also includes an upstream bacterial attachment site (attB) and a downstream SV40 polyadenylation site (SV40PA). The target gene eGFP is combined with phage attachment site (attP) and another SV40PA but without promoter. The target plasmid is co-transfected with integrase PhiC31o into the cell line. Therefore the target gene can only be expressed if integrated into the cells’ genome, and the stop-codon and SV40PA following the target gene stop the expression of bak, ensuring the survival of the cell while other cells not expressing the target gene undergo induced apoptosis.

For more details of the functional principle, please click: Jump-or-Die Functional Principle

PhiC31o will be read off because of CMV promoter. Now the integrase will combine attB and attP site and integrate construct 2 into the genome. Now the gene of interest is integrated between the tet-on promoter and bak in the cellular genome. This complex will be translated after tetracycline induction. The doubled stop codon and polyadenylation site directly after eGFP (the gene of interest) will efficiently prevent the translation of bak. Thus such cells will express the gene of interest and survive.