Biomedical Engineering Reference
In-Depth Information
Fig. 17.4.
Activation of MAP kinase pathway detected on reverse transfection array
using immunostaining. Reverse transfection arrays were produced by printing either
a control vector, v-src, or Raf-CAAX plasmids. Following the reverse transfection
process and incubation to allow for the expression of the proteins, the cells on the
arrays were fixed and stained either with
(a)
anti-phosphotyrosine antibody or
(b)
anti-phospho-Erk antibody. Slides were scanned using a GenePix4000B scanner.
Seven duplicate spots are shown from the array stained with anti-phosphotyrosine
antibody and ten duplicate spots are shown from the array stained with anti-
phospho-Erk antibody. A higher magnification image of a representative cell patch
transfected with v-src taken with a fluorescence microscope is shown to the right of
each panel
both v-src and Raf-CAAX can be detected on a reverse transfection array
using conventional immunostaining.
This system was then used to demonstrate the convenience of using co-
transfections of the pSRE-GFP reporter on reverse transfection cell arrays.
Reverse transfection arrays were printed with a mixture of Raf-CAAX and
pSRE-GFP DNA. The ratio of reporter construct to gene-of-interest con-
struct used for conventional reporter transfection experiments is typically
1:10, ensuring that each cell transfected with a reporter construct also receives
the second gene construct. To illustrate the optimal ratio for reporter trans-
fection arrays, a titration experiment was performed using various amounts
of the reporter construct pSRE-GFP and the construct encoding for Raf-
CAAX (Fig. 17.5). For establishing the position of each cell cluster within the
array, a row of 10 duplicate spots of constitutively expressed CMV-promoter
driven GFP vector (pQBI25-fPA) was printed at the top and the bottom of
the array. In between these border rows were printed spots of mixtures of
pcDNA3-Raf-CAAX vector and pSRE-GFP reporter vector at the indicated
