FIGURE 6.

pcsk7 MO injections result in erratic splicing of the pre-mRNA. A, total RNA was isolated from the control (RC MO) and different pcsk7 morphant fish and reverse transcribed into cDNA, which was amplified by PCR and run on an agarose gel. From the left, lane 1, 100-bp molecular weight marker; lane 2, pcsk7 e3 MO; lane 3, control (=RC MO with pcsk7 e3 primers); lane 4, pcsk7 e8 MO; lane 5, control (=RC MO with pcsk7 e8 primers); lane 6, 100-bp molecular weight marker. Sequencing showed that 345- and 288-bp DNA fragments represent intact, wild-type pcsk7 mRNA that can be detected in RC MO samples with the e3 (lane 3) and e8 primers (lane 5), respectively. The pcsk7 e3 MO injection resulted in three differentially sized fragments: (i) a fragment corresponding to the completely deleted exon 3 (the shortest band in lane 2), (ii) a fragment with a 58-bp deletion from the start of exon 3 followed by a 49-bp polymorphic region either from exon 3 or from exon 4 and further supplemented with the untouched end of exon 3 (the middle band in lane 2), and (iii) a faint band of wild-type exon 3 (the longest band in lane 2). The pcsk7 e8 MO injection deleted a 58-bp fragment from the end of exon 8 (lane 4) resulting in a truncated pcsk7 mRNA molecule. B, sequences for the primers used for the sequencing described in A. F, forward; R, reverse.