Modeling of IVS2 –2A>G mutation in MCP. (A) The pET (MoBiTec) exon traps cloning vectors containing wild-type or mutated genomic DNA from intron 1 (IVS1 –62) to intron 4 (IVS4 +70) of MCP. The vectors were transfected into 293T cells and the products were analyzed by RT-PCR and sequencing. Arrows 2 and 3 are the primers used for RT-PCR. Arrow 4 is the primer used for sequencing of cDNA product. MCP2, MCP3, and MCP4 indicate exons II, III, and IV of MCP. (B) Ethidium bromide–stained 1.5% agarose gel of RT-PCR products from splicing assays of transfected 293T cells. The wild-type minigene generated a product of 624 bp containing MCP exons II, III, and IV, whereas the IVS2 –2A>G mutant minigene produced a product of 521 bp lacking exon III. The wild-type sequence inserted in the reverse orientation gave a product (246 bp) that did not contain any spliced product. (C) Sequencing of cloned RT-PCR product. The RT-PCR products were subcloned into pCR2.1-TOPO and sequenced. The IVS2–2A>G mutation results in skipping of exon III. This alteration predicts a 34–amino acid loss (62-95del) in SCR2 followed by 3 amino acid changes (G96I +Y97I + Y98T) and a premature stop at L99.