Part:BBa_K4218006
The GFP-mCherry reporter system
According to previous studies, we found that exons of mitogen-activated protein kinase kinase kinase 7 (MAP3K7) was skipped in MDS patients. This mechanism gives rise to our idea of constructing the sensor based on the exon skipping of MAP3K7 to the plasmid containing reporter genes, which monitor the alteration of RNA splicing in cells. Our project will help us to evaluate the effectiveness of these sensors in diagnosing MDS patients in the future.
Usage and Biology
When the reporter was transfected into mammalian cells, the pre-mRNA of this dual reporter would be processed in either of two ways. During normal splicing of the cell, the internally shifted cryptic exon will be removed, mCherry will be expressed. Because the stop codon was located in front of the GFP gene, GFP could not be translatable and red fluorescent could be detected in cells. In the case where splicing is inhibited, the internally shifted recessive exon will not be removed, the mCherry gene is shifted. The GFP is expressed, and the cells show green fluorescence. Therefore, we can monitor the level of splicing in the cell through detecting the different color of fluorescence.
Name: MAP3K7-GFP
A previous study indicated exons of MAP3K7 were skipped in MDS patients. In order to detect the dysregulation of RNA splicing, we designed two exon-skipping LUC reporters (MAP3K7-LUC) which contains parts of the MAP3K7 intronic and exonic sequences in the luciferase gene (more details about the principle were listed in the project description and the working model was listed below).
Results
1.Construction of MAP3K7-GFP
In order to visualize the situation of RNA splicing, we designed a new fluorescent plasmid sensor. The plasmid of MAP3K7-GFP was synthesized by Nanjing Genscript Biotechnology Corporation. MCherry gene was fused with a GTGAGT-recessive exon-CCACAG mingene. And a shifted in-frame termination codon (TGATG) was inserted between GFP and mCherry. The fused genes were respectively inserted into the pcDNA3.1(+)-C-eGFP plasmid vector which digested by BamH Ι and EcoR V (Fig 1). When we got these plasmids, we transformed them into DH5α bacteria and screened the positive clones on LB solid plates containing ampicillin (Fig 2). To validate these plasmids, MAP3K7-GFP was digested with BamH Ι and EcoR V and analyzed on 1% Agarose Gel (Fig 3).
1.Functional verification of MAP3K7-GFP plasmid sensors.
Using the same method, we tested the ability of MAP3K7-GFP to detect dysregulation of RNA splicing in 293T cells with or without Pladienolide B treatment. The plasmid MAP3K7-GFP was transfected into 293T cells. PB (final concentration: 0.3 ng/μl) was added to disturb the process of RNA splicing. According to the principle of our plasmid sensor, during normal splicing of the cell, mCherry will be expressed but GFP could not be translatable and red fluorescent could be detected in cells. In the case where splicing is inhibited, the GFP is expressed, and the cells show green fluorescence (more details about the principle were listed in the project description). After 48 h cell culturing, we observed the fluorescence expression of cells under fluorescence microscope (10x eyepiece, 20x objective). We found that the group transfected with plasmid only had more red fluorescence than the group treated with PB. After transfection with MAP3K7-GFP plasmid, the GFP fluorescence was relatively less and dark. The GFP fluorescence was significantly enhanced in cells treated with PB, indicating that the inhibition of cell splicing was well detected by the plasmid sensor(Fig 4). After the observation of the cells, we digested them and added into a 96-well plate, and the fluorescence values of mCherry and GFP were measured by microplate reader (SpectraMax i3). We used the ratio of fluorescence values of mCherry and GFP to measure the inhibition of cell splicing. We found that the ratio of mCherry and GFP was 0.73 in cells transfected with MAP3K7-GFP. And the ratio of mCherry and GFP reached to 0.16 in cells following PB treatment (Table 1, Fig 5). There results were also consistent with the images of fluorescence in Fig 16. In conclusion, MAP3K7-GFP plasmid sensor can monitor the level of splicing in the cell well by detecting the different color of fluorescence.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1126
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1126
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1126
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1126
- 1000COMPATIBLE WITH RFC[1000]
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