Journal: BIO-PROTOCOL
Article Title: Assaying the Effects of Splice Site Variants by Exon Trapping in a Mammalian Cell Line
doi: 10.21769/bioprotoc.2281
Figure Lengend Snippet: Figure 1. Exon trapping assay Vector exons V1 and V2, are depicted as black boxes and TEK exons 5, 6, and 22 are shown in gray. Vector exon-specific primers are indicated by half-arrows in (A) and (B). Wild-type (WT) and mutant (M) splicing products, with included exon sizes in base pairs, are indicated by dashed lines above and below the construct, respectively. The locations of the splice site mutations are shown as an asterisk (*). A. Wild-type (WT-5) and mutant (M-5) genomic fragments containing TEK exons 5 and 6 were used to model the c.760+2T>C mutation. B. Wild-type (WT-22) and mutant (M-22) genomic fragments containing TEK exon 22 were used to model the c.3300+2delT mutation. C. Gel electrophoresis of RT-PCR products from transfected COS-7 cells. ‘Empty Vector , cells transfected with vector containing’ no gDNA insert; ‘TF –ve’ (transfection negative), cells transfected with QIAGEN buffer EB only; ‘PCR –ve’ (PCR negative), PCR contamination control substituting water for cDNA template. Wild-type and mutant transcript content, determined by Sanger sequencing, is depicted to the right of the gel image. The additional 21 bp of intron 5 sequence identified within the M5 transcript is shown incorporating a premature termination codon between exons 5 and 6.
Article Snippet: Gel electrophoresis tank (and tray) (Bio-Rad Laboratories, model: Wide MiniSub GT Cell) 16.
Techniques: Plasmid Preparation, Mutagenesis, Construct, Nucleic Acid Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Transfection, Control, Sequencing
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