Biomedical Engineering Reference
In-Depth Information
fected with pCMV-GFP, where the GFP expression is driven by the strong
CMV promoter. In addition, the absolute number of cells co-transfected with
the SRE-GFP reporter was somewhat lower than the number of cells that
were transtected by the single pCMV-GFP plasmid. Nonetheless, the GFP
signal generated by gene-specific activation of the SRE promoter was easily
detectable above the background signal using both laser scanning (Fig. 17.5a)
and fluorescence microscopy (Fig. 17.5b). The highest reporter signal was seen
in the co-transfections using a relatively high pSRE-GFP reporter concentra-
tion compared to conventional transfections (Fig. 17.5b). Thus, the optimal
range of pSRE-GFP and co-transfected gene-of-interest is 0.025-0.10
g/
L
µ
µ
and 0.01-0.05
L, respectively. A key advantage of using the GFP reporter
system, as illustrated in Fig. 17.5, is that SRE activation can be assessed
and quantitated in unfixed, unprocessed cells. The reverse transfection array
shown was imaged without fixing the cells. Instead, media was removed and
the array was covered with a coverslip and imaged immediately. A substan-
tial time savings was afforded using this reporter method compared to the
immunofluorescent staining method described in Fig. 17.4.
Thus, this chapter outlines the use of reporter constructs to monitor the
activity of proteins produced by reverse transfection. We have demonstrated
the utility of this technique using a model MAP kinase system. The sim-
plicity and convenience of this reporter co-transfection method for reverse
transfection arrays will be especially appealing for high throughput screening
applications where post-transfection processing would be cumbersome and
prohibitive. This method could be extended to larger reverse transfection ar-
rays used for screening genes of unknown function simply by including the
reporter construct in the gelatin printing solution. In addition, the develop-
ment of other reporter systems that are more quantitative than GFP and are
still suitable for array applications would make reverse transfection reporter
systems even more attractive.
g/
µ
µ
17.3 Reagents and Protocols
•
Gelatin, Type B: 225 Bloom (Sigma #G-9391)
GAPS
TM
slides (Corning #2549)
•
•
CMP3, CMP7, and CMP10B Micro Spotting Pins (Telechem Interna-
tional, Inc.)
•
PixSys 5500 Robotic Arrayer (Cartesian Technologies, Model AD20A5)
•
CoverWell Incubation Chambers (Grace BioLabs #PC200)
•
QuadriPerm chambers (Sigma)
•
Effectine reagent (Qiagen #301425)
•
pQBI25-fPA encoding for GFP (Qbiogene)
•
pcDNA3-v-src, pcDNA3-Raf-CAAX were kindly provided by Dr. Steve
Martin.
•
pcDNA3-HA-KRasV12 was kindly provided by Dr. Steve Taylor.
