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. 2018 Oct 30;3(1):201–221. doi: 10.1210/js.2018-00130

Figure 2.

Figure 2.

An in vitro assay revealed aberrant splicing of variants c.940G>A, c.990G>A, and c.1173C>T. (A) Minigene construction. Diagram of exon 5 and parts of flanking introns 4 and 5, inserted into the MCS of the pET01 construct (Lower). In the pET01 construct, the intron containing the MCS is flanked by the 5ʹ-donor and 3ʹ-acceptor splice sites of preproinsulin 5ʹ and 3ʹ exons, respectively (green arrows) (http://www.mobitec.com/cms/products/bio/04_vector_sys/exontrap.html). The expression of this vector sequence was driven by the promoter present in the long terminal repeat of Rous sarcoma virus, followed by a short stretch of a eukaryotic gene (phosphatase). The sequences containing the mutations detected in CYP11A1 exons 5 and 7 (mutant) or those that did not (WT) were cloned into the MCS of pET01. The primers used in the RT-PCR experiments within the preproinsulin 5ʹ and 3ʹ exons are indicated by the black arrows (Supplemental Table 5). (B) Representative results of RT-PCR analysis using HEK293 cells transfected with an empty pET01 vector (empty vector), the pET01 vector containing the WT exon or the mutant exon: (Left) the c.940A in exon 5, (Middle) the c.990A change in exon 5, (Right) the C.1174T change in exon 7. A transcript of 225 bp was observed in the empty and mutant vector RT-PCRs, for all variants investigated, corresponding to the two-exon amplification product resulting from splicing of the preproinsulin 5′ and 3′ exons from the vector. For WT vectors containing c.940G and c.990G, a 386 bp transcript was observed corresponding to the three-exon amplification product resulting from correct splicing of CYP11A1 exon 5 between the two vector exons. The intermediate band for exon 5 constructs, at ~350 bp (asterisk), was shown to be a mixture of a sequence that included both 386- and 225-bp bands. For WT c.1173C, a 304-bp transcript corresponding to the size of the three-exon amplification product resulting from correct splicing of CYP11A1 exon 7 was observed. Sanger sequencing confirmed these findings (data not shown).