Minigene and Expression vectors design
CFTR minigenes were created by cloning a sequence composed by an
~150 bp intronic regions flanked by the exon examined,
respectively exon 5, 9, 13, 16 in the previously described pTB minigene
(Pagani et al., 2003) or exon 18 in pFAN minigene (Mattioli et al.,
2014). Mutations were introduced in the WT of the respective minigene by
site-directed mutagenesis using the Quick-Change Site-directed
Mutagenesis Kit II (Agilent, Santa Clara, USA). Exon 13-Δ minigenes were
created by overlapping PCR, using specific oligonucleotides
complementary to up-downstream sequences of the deleted region in a
pTB-NdeI minigene. ExSpeU1s were created by replacing the sequence
between the BclI and BglII sites with oligonucleotides as previously
reported (Pagani et al., 2002). Splicing competent exon 13 minigene and
its mutants were made from cDNA cloned in the vector pcDNA 3.1, while
for exon 16 we used the described minigenes (Igreja et al., 2015). The
identity of minigene constructs was ultimately confirmed through
sequencing analysis.