Biomedical Engineering Reference
In-Depth Information
Fig. 17.2.
Activation of pSRE-GFP reporter by mutationally-activated MAP ki-
nase pathway signaling proteins. The pSRE-GFP reporter construct was generated
by linking the green fluorescence gene to the SRE enhancer element. Conventional
co-transfection experiments were performed using pSRE-GFP along with DNAs
encoding for three activated mutant signaling proteins, v-src, RasV12, and Raf-
CAAX in HEK293 cells. Following 48 hours, GFP-producing cells were visualized
using fluorescence microscopy
tration of 0.2%). DNA/gelatin solutions were printed in an array format on
Corning GAPS
TM
slides using a Cartesian PixSys 5500 printer. The printed
slides were dried in a vacuum dessicator for two hours. Effectine transfection
reagent for each slide was prepared by mixing 150
µ
lECBuffer,16
µ
l En-
hancer, and 25
l Effectine transfection reagent in a 1.5 ml micro-centrifuge
tube. This solution was added to a CoverWell Incubation Chambers (Grace
BioLabs catalog #PC200) and the slide was pressed down onto the CoverWell
Chamber, sealing the transfection reagent between the slide and the chamber.
Incubation of the array with the transfection reagent between 15-20 minutes
is optimal. Following the incubation, the CoverWell was peeled off the slide,
excess reagent removed from the slide, and the slide was placed in a Quad-
Perm cell culture device. During the Effectine incubation, HEK293 cells were
prepared as follows. HEK293 cells grown in T75 flasks were trypsinized, resus-
µ
