In vivo minigene splicing. (A) For the in vivo minigene splicing studies, an additional probe was constructed. To generate CA0, having no CA repeats, a BamH1/BamH1 segment representing nucleotides 186 to 489 was excised from the CA21 probe. The resulting construct lacks the CA repeat region at nucleotide 250 to 291. (B) B16F10 cells were transfected with 2 μg pcDNA3.1(–) containing either the CA6, CA21, or CA0 cassettes. Total RNA was then extracted at 2, 3, 4, and 5 hours after transfection. Spliced mRNA was amplified by RT-PCR, and separated by agarose gel electrophoresis. The amount of spliced mRNA (open arrowhead) was calculated using image J³⁸  and is expressed as the percentage of total mRNA (nonspliced plus spliced mRNA). (C) The percentage of spliced mRNA is plotted as a function of time (hours) after transfection with the minigene cassette. The graph depicts the levels obtained with the CA21 (•), CA6 (♦), or CA0 (X-X) minigene cassettes. Each data entry represents the mean of triplicate measurements in a single experiment that is representative of 4 independent experiments.