Data in support of a functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II

Abstract

This data article contains insights into the methodology used for the analysis of three exonic mutations altering the splicing of the IDS gene: c.241C>T, c.257C>T and c.1122C>T.We have performed splicing assays for the wild-type and mutant minigenes corresponding to these substitutions. In addition, bioinformatic predictions of splicing regulatory sequence elements as well as RNA interference and overexpression experiments were conducted.The interpretation of these data and further extensive experiments into the analysis of these three mutations and also into the methodology applied to correct one of them can be found in “Functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II” Matos et al. (2015) [1].

Functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II

“…Using the Rapid Ligation Kit (Roche Applied Science, Indianapolis, USA) they were also cloned into pcDNA3.1-myc, a modified plasmid vector (Invitrogen, Carlsbad, USA) that contains the sequence for the Myc epitope (to be used as a sequence tag) upstream of the multiple cloning site (Fig. 1A in [34]). …”

“…Since an IDS pseudogene [3][4][5] exists that shows high homology with the IDS gene in the region corresponding to exons 2, 3 and introns 2 and 3, primers for these amplifications were designed to anneal at intron 1 and 3 in such a way that the amplified fragment matched the corresponding gene and not the pseudogene. A BamHI (Forward primer -Intron 1 BamHI F) and XhoI (Reverse primer -Intron 3 XhoI R) restriction enzyme cleavage site tail was added to each oligonucleotide (Table I in [34]). The PCR conditions were as described above.…”

“…After subcloning into the TOPO vector, each insert was excised with EcoRI, purified, and cloned into the pSPL3 final vector ( Fig. 1C in [34]). All clones were verified by DNA sequencing.…”

“…is flanked by functional splice donor and acceptor sites. For this, gene fragments comprising exon 8 and its intronic flanking regions were amplified from the DNA of both control and patient fibroblasts using the primers shown in Table I [34], employing the PCR conditions described above. After subcloning into the TOPO vector, each insert was excised with EcoRI, purified, and cloned into the pSPL3 final vector ( Fig.…”

“…Total mRNA was isolated using the High Pure RNA Isolation Kit (Roche Applied Science, Indianapolis, USA) and reverse-transcribed using the "Ready-To-Go You-Prime First-Strand Beads" Kit (GE Healthcare, Buckinghamshire, UK), following the manufacturer's protocol. For RT-PCR, the specific primers shown in Table I [34] were designed using the Ensembl database (www.ensembl.org; Transcript: IDS-001 ENST00000340855). cDNA amplifications were performed in a total volume of 25 μl using the Hot-Start PCR Mastermix Immomix Red Kit (Bioline, London, UK), following the manufacturer's instructions, employing 0.2 μM of each primer and an annealing temperature of 60°C (35 cycles).…”