Cell Cycle Arrest Assay
Example of Application in RB field: RB-dependent block of cell cycle at G1 restriction point
Over-expressed pRB is able to repress E2F-driven activation of target genes. Promoters of E2F target genes often contain E2F-binding sites which serve as enhancer elements as they recruit the E2F transcription factors, which subsequently recruit subunits of the RNA polymerase II holoenzyme. A large subset of E2F targets, collectively referred to as "E2F-cell cycle targets" (ex. Cyclin E, PCNA, MCM4, etc.) work to bypass the pRB-mediated G1 arrest, to push the cell cycle into S-phase, thus committing the cell toward complete DNA replication unless interrupted by genotoxic insult such as DNA damage.
Principle
To assess cytostatic potential of pRB mutants, pRB expression vectors are transfected into RB-negative cells (ex. Saos-2 cells). By measuring the % of cells in G1 phase, one can determine how effective pRB or the pRB mutant is at conferring a G1 arrest. Often, CD20 is co-transfected to serve as a marker for successful transfection. In this case, only cells which have been successfully transfected are used in the analysis.
How is G1 measured?
Over-expressed pRB is able to repress E2F-driven activation of target genes. Promoters of E2F target genes often contain E2F-binding sites which serve as enhancer elements as they recruit the E2F transcription factors, which subsequently recruit subunits of the RNA polymerase II holoenzyme. A large subset of E2F targets, collectively referred to as "E2F-cell cycle targets" (ex. Cyclin E, PCNA, MCM4, etc.) work to bypass the pRB-mediated G1 arrest, to push the cell cycle into S-phase, thus committing the cell toward complete DNA replication unless interrupted by genotoxic insult such as DNA damage.
Principle
To assess cytostatic potential of pRB mutants, pRB expression vectors are transfected into RB-negative cells (ex. Saos-2 cells). By measuring the % of cells in G1 phase, one can determine how effective pRB or the pRB mutant is at conferring a G1 arrest. Often, CD20 is co-transfected to serve as a marker for successful transfection. In this case, only cells which have been successfully transfected are used in the analysis.
How is G1 measured?