Figure 3

The PITX2c minigene is expressed and the mRNA splices normally. A. HEK293 cells were transfected with a GFP transfection control plasmid with (+) or without (-) 2 μg of the minigene. Transfected-cell RNA was isolated and subjected to RT-PCR with primers to the minigene (top) or GFP (bottom) and the products were resolved on an agarose gel. The position of correctly spliced product for the minigene and GFP is shown at the right. M, 100 bp size standards with selected sizes shown. B. Human cornea stromal cells were either mock transfected (-) or transfected with 2 μg of minigene DNA (+). RNA was isolated and analyzed by RT-PCR with primers that amplify only minigene mRNA (top) or those that amplify both minigene and endogenous PITX2c mRNA (middle), or to cellular GAPDH (bottom), and products were resolved on an agarose gel. Primers are depicted in Figure 2B. M, 100 bp size standards with selected sizes shown. The positions of correctly spliced products are shown at the right. C. Sequence of spliced products. RT-PCR products were cloned and sequenced. At the top is a schematic of the pre-mRNA with exon sequences boxed and in uppercase and intron sequences in lowercase. Lines extend from splice sites to the spliced product sequence shown at the bottom.