Pet01 exontrap vector

A fragment of the human IRF6 gene, comprising exon 7 (of nine) and 309 bp of intron 6 and 428 bp of intron 7, was amplified by polymerase chain reaction (PCR) using HiFi HotStart DNA Polymerase (Kapa Biosystems) and human genomic DNA isolated from an unaffected control individual with the following primer pair containing restriction site linkers: hIRF6-I6 forward (5′-CAATCTCGAGCCGACTCAGTCAG TATAGCGTGGG-3′; XhoI site underlined) and hIRF6-I7 reverse (5′-AGCTCTAGATGTAACTCCCTTC TTTGTTGCC-3′; XbaI site underlined). The purified product was double digested with XhoI and XbaI and cloned into a similarly digested pET01 exon trap vector (MoBiTec, Goettingen, Germany) to generate the wildtype (WT; c.921C) construct.
Plasmids for the expression of Splice Regulatory Factor 1 (pcDNA-FLAG-SF2; Addgene 99021) and Splice Regulatory Factor 2 (pcDNA3.1-SC35-cMyc; Addgene 44721) were separately co-transfected with the empty pET01 vector, or either the WT (c.921C) or mutant (c.921T) vectors into COS7 cells and total RNA was extracted two days post transfection (n = 6 per group), as described above. Following cDNA synthesis, PCR products were amplified as previously described, separated on a 1.2% agarose gel and quantified as above.

To determine the effect of the ATP11A c.3322_3327+2dupGTCCAGGT variant, we performed a minigene splicing assay. A 554-bp genomic sequence, including exon 28 and flanking introns, was PCR amplified from wildtype and heterozygous DNA samples using specific primers introducing XhoI and BamHI restriction sites. After digestion, PCR amplicons were cloned into the pET01 Exontrap vector (MoBiTec) using LigaFast DNA ligation kit (Promega). Wildtype and mutant minigene constructs were transfected into HEK293T cells, and RNA was extracted 48 h post-transfection using Trizol reagent. The synthesized cDNA was amplified using primers complementary to the 5′ and 3′ exons of the Exontrap vector. RT-PCR products were then visualized on gel, purified, and Sanger sequenced.

Genomic DNA from WT and ∆IDR prestin mouse tails was amplified for the regions flanking exons 17-18 (+150 bp before and after) and cloned into a pET01 exon-trap vector (MoBiTec, Göttingen, Germany) containing a polylinker or multiple cloning site (MCS) in between two exons with donor and acceptor sequences. The day before transfection, HEK293T cells were seeded on a 6-well plate so that they were 60–70% confluent at the time of transfection. The pET01 constructs were introduced using Effectene (Qiagen) following the manufacturer’s directions. Total RNA was isolated from each well using an Absolutely RNA miniprep kit (Agilent) and cDNA synthesis was carried out with M-MLV-RT (Promega) following the recommended protocols. An pET01-specific primer (cDNA primer 1, 5′- GATCCACGATGCCGC -3′) was used for the reaction. PCR was performed using GoTaq Flexi DNA polymerase with DNA primer pairs 2 (5′- GATCTGCTTCCTGGCCC-3′) and 3 (5′- GGCCACCTCCAGTGCC -3′) from the exon-trap protocol^{33 (link)}.