Difference between revisions of "Part:BBa K4218004"

Latest revision as of 01:24, 12 October 2022

The dual luciferase 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

To measure the alteration of RNA splicing, we engineered one dual luciferase reporter system where two luciferase genes were fused with GTGAGT-recessive exon MAP3K7-CCACAG minigenes . Upon transfection of the dual luciferase reporters into mammalian cells, the pre-mRNA of this dual reporter would be processed in either of two ways. During normal splicing in cells, the internal stop codon would be removed, then the upstream reporter gene (Firefly luciferase, Fluc) and the downstream reporter gene (Renilla luciferase, Rluc) will be placed in the same reading frame to generate a fusion protein Fluc-Rluc. In the case that splicing in inhibited by splicing inhibitors, the stop codon in the recessive exon would lead to the translation termination of the mRNA, thus producing the Fluc protein alone. Therefore, the Fluc gene is expressed regardless of whether splicing occurred, whereas the downstream Rluc gene can only be expressed after splicing.

Results

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).

1.Construction of MAP3K7-LUC

The plasmid of MAP3K7-LUC was synthesized by Nanjing Genscript Biotechnology Corporation. The map of the plasmid was listed below (Fig 1). When we got the plasmid, we transformed it into DH5α bacteria and screened the positive clones on LB solid plates containing ampicillin (Fig 2). To test the quality of the plasmid, MAP3K7-LUC was analyzed on 1% Agarose Gel (Fig 3). Moreover, MAP3K7-LUC was also confirmed by using sequencing (Fig 4).

2.Functional verification of the MAP3K7-LUC plasmid sensor

In order to test the ability of MAP3K7-LUC to detect dysregulation of RNA splicing, Pladienolide B (PB, an RNA splicing inhibitor) was used. The plasmid MAP3K7-LUC was transfected into 293T cells, respectively. PB (final concentration: 1 ng/μl) was added to disturb the process of RNA splicing. According to the principle of our plasmid sensor, the fusion proteins (Fluc-Rluc) were expressed in 293T cells. However, when RNA splicing was interrupted, only the Fluc proteins were induced (more details about the principle were listed in the project description). After transfection, cells were lysed and the expression of luciferase was measured by using plate reader (SpectraMax i3). In normal 293T cells, the expression of the fusion proteins (Fluc-Rluc) was high (Table 1, Fig 5). PB treatment induced the downregulation of the fusion proteins (Table 1, Fig 5). The ratio of (Rluc+ Fluc) to Rluc intensity [(Rluc+Fluc)/Rluc] was significantly decreased in cells treated with PB, compared with normal cells (Table 1, Fig 5). These results suggested MAP3K7-LUC sensor can detect the alteration of RNA splicing in cells.

3.The sensitivity of MAP3K7-LUC sensor

In order to test the sensitivity of MAP3K7-LUC sensor, different concentrations of plasmid (final concentration: 1 ng/μl、0.6 ng/μl、0.2 ng/μl) were transfected into cells. PB treatment decreased the ratio of (Rluc+ Fluc) to Rluc intensity [(Rluc+Fluc)/Rluc] in cells transfected with MAP3K7-LUC plasmid. Our data indicated even a small number of plasmid sensors can detect the changes of intracellular splicing, suggesting the sensitivity of our sensors is high (Table 2 and , Fig 6).

4.The effect of the MAP3K7-LUC sensor as the monitor to detect the alteration of RNA splicing.

To test the effect of the MAP3K7-LUC sensor as the monitor to detect the alteration of RNA splicing, cells were transfected with plasmid sensors following treated with different concentration of PB (0.6 ng/μl, 0.2 ng/μl). The treatment with different concentration of PB represents the different degrees of RNA splicing alteration. In the presence of PB, the luminescence activity was significantly decreased in a dose dependent (Table 5 and 7, Fig 9 and 11). Moreover, based on the luciferase values in cell treated with different concentration of PB, the standard curve of MAP3K7-LUC sensor was made by EXCEL (Table 3 and 4, Fig 8 and 9). We find that the value of luciferase is dependent on the concentration of PB in cells. Based on the formula, the correlation coefficient (R2 value) of MAP3K7-LUC was 0.9915.

Sequence and Features