What are the differences between the phenotypic effects of the GAPDH and EG5 positive controls for pGIPZ?
Between EG5 and GAPDH, only EG5 causes a change in phenotype. This was seen by silencing of the human EG5 (KIF11) gene in HEK293T cells (data shown in the pGIPZ technical manual: http: //dharmacon. horizondiscovery. com/uploadedFiles/Resources/gipz-lentiviral-shrna-manual. pdf. The cells were transduced using an EG5 lentiviral shRNAmir and stained for DNA (DAPI, blue), tubulin (anti-tubulin, green), and EG5 (anti-EG5, red) 48 hours later. EG5 knockdown results in disruption of normal cell division and causes the formation of half spindles. Cells transduced with EG5 shRNA are arrested in mitosis and show monoastral microtubular arrays. By contrast, control cells show normal bipolar spindles and microtubule networks in mitosis and in interphase (cell on the bottom left). GAPDH should not affect the cellular phenotype or proliferation rate except as an off target effect. However, if GAPDH were to be knocked down completely, it would likely kill the cells.