Cloning and Mutagenesis of ABCA4 Minigene

pIC-neo.Rho3-5.MCS⁶³ (link) was generated by replacing the USH2A sequences of pCI-neo.Rho.USH2A-PE40-wt by a custom MCS containing the following restriction enzyme recognition sites: XhoI, EcoRI, MluI, EcoRV, XbaI, SalI, and Cfr9I designed to aid in downstream cloning steps. The custom MCS was generated by annealing DNA oligonucleotides 5′-CTCGAGAATTCACGCGTGGTGATATCACCTCTAGAGTCGAC-3′ and 5′-CCCGGGTCGACTCTAGAGGTGATATCACCACGCGTGAATTCT-3′. The resulting fragment was used in a ligation mixture together with the backbone plasmid, digested with XhoI and Cfr9I (Thermo Fisher Scientific, Waltham, MA, USA).
To generate a ABCA4 c.5461-10T>C minigene, the pCI vector backbone and a synthetic dsDNA sequence (gBlock; Integrated DNA Technologies, Coralville, IA, USA) containing the ABCA4 minigene genomic region with exon 39 and parts of adjacent introns (1:94,477 466–94,476 525, GRCh37) (Figure S1A) and the c.5461-10T>C mutation were digested using the EcoRI (New England Biolabs, Ipswich, MA, USA) and SalI (Thermo Fisher Scientific). The digested vector was loaded on 1% agarose gel, isolated and purified using the Nucleospin Gel and PCR Clean-up kit according to the manufacturer’s instructions (MACHEREY-NAGEL, Düren, Germany). The digested gBlock was purified directly using the same kit. Digested fragments were ligated overnight at 16°C with T4 ligase (Thermo Fisher Scientific) following the manufacturer’s protocol. The ligation reaction was used to transform DH5α-competent cells (Thermo Fisher Scientific) according to manufacturer’s protocol.
To generate an ABCA4 wild-type midigene (construct with >1 exons and surrounding introns), genomic DNA from HeLa cells (ATCC, Manassas, VA, USA) was extracted with the DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany). The ABCA4 genomic region between intron 37 and intron 41 (1:94,482 094–94,473 896, GRCh37) (Figure 2A) was amplified with primers (Integrated DNA Technologies) containing recognition sites for EcoRI and SalI, using the Phusion High-Fidelity DNA Polymerase kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. The wild-type PCR product and the digested pCI vector backbone were ligated and transformed into GT115 competent cells (InvivoGen, San Diego, CA, USA). To introduce the ABCA4 c.5461-10T>C mutation, the wild-type plasmid and a gBlock (Integrated DNA Technologies) from intron 37 to intron 41 containing the c.5461-10T>C mutation were digested with BoxI and BsiWI (Thermo Fisher Scientific) and subsequently ligated and transformed into GT115-competent cells.