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. 2012 Dec 7;288(3):1750–1761. doi: 10.1074/jbc.M112.397604

FIGURE 4.

FIGURE 4.

A–D, Bma-pdi-2 introduced into a C. elegans pdi-2 null mutant results in complete repair of the mutant phenotypes. A, wild type C. elegans N2 strain; B, C. elegans pdi-2 homozygote mutant (from a heterozygous mother); C and D, C. elegans pdi-2 homozygote mutant carrying Bma-pdi-2 on an extrachromosomal array with D showing GFP expression from the transformation marker dpy-7prom::gfp carried on the same extrachromosomal array. Scale bars represent 100 μm. E, genotyping by single-worm PCR. A set of three primers designed around the Cel-pdi-2(tm0689) deletion were use to genotype all transgenic lines to ensure mutant homozygosity. The upper 320-bp band identifies the deleted region, and the 185-bp band indicates the wild type. The three transgenic lines (Ex1–Ex3) generated for each construct, Bma-pdi-2 genomic and Bma-pdi-2 cDNA + synthetic intron (SI), all produce only the deletion sized amplicon. Lane 7, the larger deletion-derived amplicon and smaller wild type-derived amplicon from a pdi-2(tm0689) heterozygote (Het). Lane 8 is wild type (WT), and lane 9 represents no template control (NT). F, RT-PCR showing expression of Bma-pdi-2 in transgenic C. elegans. Expression is shown for three transgenic lines (Ex1–Ex3) for each Bma-pdi-2 construct and Bma-pdi-2 genomic and Bma-pdi-2 cDNA + synthetic intron (SI). For the Bma-pdi-2 genomic construct, primers amplifying the full-length Bma-pdi-2 sequence were used. Lanes 1–3 show expression of the 1512-bp spliced Bma-pdi-2 cDNA in lines carrying the genomic construct. Lane 4, the 3579-bp amplicon generated using the Bma-pdi-2 genomic plasmid (P) as template. Lane 5, no template (NT) control. For the Bma-pdi-2 cDNA + synthetic intron (SI) construct, primers corresponding to Bma-pdi-2 sequences situated either side of the 51-bp synthetic intron were used. Lanes 6–8 show the 486-bp spliced amplicon derived from three transgenic C. elegans lines compared with the 537-bp plasmid-derived product (P, lane 9) and no template (NT) (lane 10). Bma-pdi-2 from genomic construct; spliced, 1512 bp; unspliced, 3579 bp. Bma-pdi-2 from construct with cDNA and a synthetic intron; spliced, 486 bp; unspliced, 537 bp.