3.3 DMD mRNA expression in the transfected cell lines
The minigene splicing assay was to determine whether this genetic variant could result in aberrant splicing during gene expression. 293T and MCF-7 cells were transfected and a total of 8 samples were collected after 48h of transfection.
3.3.1 pcMINI-DMD -wt/muttranscriptional analysis results
A schematic diagram of the pcMINI-DMD -wt/mut construct is shown in Figure 3A. RT-PCR obtained the expressed gene inserts from the pcMINI-DMD-wt/mut vector in 293T and MCF-7 cells. The different splicing products for wild-type (band a: wt lane, 477 bps) and variant type (band b: mut lane, 470 bps) are obtained on electrophoresis gel with close bp size (Figure 3B). Sequencing results indicated that the minigene of wild-type (from pcMINI-DMD-wt) formed mRNA containing complete exon 19 (Figure 3C, D). For the expressed minigene from pcMINI-DMD-mut, the intron c.2293-1G>C caused aberrant splicing, leading to the absence of 7 bp on the left side of exon 19 (Figure 3C, D).
3.3.2pcMINI- C- DMD -wt/mut transcriptional analysis results
We further determined that mutation c.2293-1gG> C could affect the normal splicing on mRNA of DMD gene and the deletion of 7bp on the left side of Exon19 was seen in the pcMINI-C- DMD -wt/mut vectors (Figure 4A, B, C, D).
Through in-vitro Mini-gene splicing assay, we confirmed that the biofunction of the novel splicing mutation (c.2293-1gG> C). This gene variant is responsible of causing a frame shift of the transcripts, which could lead to the generation of an abnormal dystrophin protein.