A genomic fragment containing the WT sequence corresponding to each variant and its surrounding exons and introns was amplified from HEK293T cells by PCR with specific primers (Table S4 ) and inserted into the pSPL3 vector (Life Technologies). Each variant was reintroduced by site-directed mutagenesis. COS-7 cells were transfected with the resulting plasmids in the presence of Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific). Cells were harvested 24 h after transfection, and total RNA was extracted with the RNeasy Mini Kit (QIAGEN). Reverse transcription was performed with the iScript Reverse Transcription Supermix (Bio-Rad), according to the manufacturer’s instructions. After cDNA synthesis, PCR was performed with the SD6 (5′-TCTGAGTCACCTGGACAACC-3′) and SA2 (5′-ATCTCAGTGGTATTTGTGAGC-3′) primers. The amplified cDNAs were inserted into the pCR4-TOPO vector (Thermo Fisher Scientific) and subsequently sequenced with the M13 forward (5′-GTAAAACGACGGCCAG-3′) and M13 reverse (5′-CAGGAAACAGCTATGAC-3′) primers. For each variant, we analyzed 100 colonies, to determine the ratio of spliced products from WT and mutant minigenes.
Pspl3 vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PSPL3 vector is a plasmid-based DNA expression vector designed for the production of recombinant proteins in bacterial host cells. The vector features a strong bacterial promoter, a multiple cloning site for the insertion of the gene of interest, and a selectable antibiotic resistance marker for the selection of transformed cells.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Iscript reverse transcription supermix, by Bio-Rad (1 mentions)
Pcr4 topo vector, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Pgem t vector, by Promega (1 mentions)
Quikchange lightning kit, by Agilent Technologies (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Quickchange 2 xl kit, by Agilent Technologies (1 mentions)
Cdna synthesis, by Qiagen (1 mentions)
Nucleospin rna kit, by Qiagen (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Ta cloning vector, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Polyjet reagent, by SignaGen (1 mentions)
7 protocols using pspl3 vector
1
Minigene Splicing Analysis of Variants
2
In vitro Splicing Profiling of PTS Variants
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For in vitro evaluation of the splicing profile different minigene constructs were used. The previously described splicing reporter plasmid encompassing PTS exons 2–4 cloned in the pCR3.1 vector [26 (link),28 (link)] was used as template for deep intron 2 variants c.164-672C>T and c.164-716A>T. The pSPL3 vector (Life Technologies) was used in the analysis of the c.243 + 3A>G variant. Using this vector, we cloned PTS exon 4 and ∼150 bp of the flanking intronic regions, amplified from genomic DNA using specific primers (Forward: 5′-GCTTCCATGCTGAGGTCAAT-3′ and Reverse: 5′-ACTATTCCCCAACACCCACA-3′). Gene fragments were cloned into the pGEMT vector (No. A1360; Promega). The insert was excised with EcoRI and subsequently cloned into pSPL3. Variant minigenes containing the desired nucleotide changes were generated by site-directed mutagenesis with QuikChange Lightning Kit (Agilent Technologies, Santa Clara, CA, USA) using primers introducing the change and their reverse complement. Sanger sequencing confirmed the identity of the constructs.
3
In vitro minigene assay to investigate TAPT1 splicing defect
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To confirm the potential role of TAPT1 deep intronic mutation (c.1237‐52 G>A) in splicing defect, an in vitro minigene assay was done using pSPL3 exon trapping vector (Westin et al, 2021 (link); Iturrate et al, 2022 (link); Rodriguez‐Muñoz et al, 2022 (link)). A genomic DNA fragment from patient cells (V.1(F1)) containing TAPT1 exon 12 flanked by 200 bps upstream and 500 bps downstream intronic sequences were cloned into pSPL3 vector (Invitrogen) using EcoR1/BamH1 restriction sites. Subsequently, the mutant construct was used as a template to generate a rescue construct by introducing c.1237‐52 A>G change using QuickChange II XL kit (Agilent). Both mutant and rescue constructs were verified by direct Sanger sequencing. Then, HEK293T cells were transfected with 4 μg of DNA (pSPL3‐c.1237‐52 G>A, pSPL3‐rescue or empty pSPL3 as a control) using Opti‐MEM (Gibco) and Lipofectamine 3000 reagent (Invitrogen). Total RNA was extracted 24 h after transfection by NucleoSpin RNA kit and 3 μg of RNA was used for cDNA synthesis (Qiagen). To compare the splicing patterns of cells transfected with different constructs, RT–PCR was performed using vector‐specific primers (SD6 and SA2). The PCR products were loaded on 2% agarose gel and purified after gel extraction. The transcripts were analyzed by direct Sanger sequencing. Primer sequences are shown in Table EV1 .
4
Evaluating NCF4 Splicing with Mini-genes
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To evaluate the in vitro splicing with mini-genes, we amplified a 178 bp genomic fragment spanning the parts of intron 9 and exon 10 of the NCF4 gene from bovine genomic DNA. After digestion with EcoRI and XhoI, the segment with the wild-type AA or the mutant type GG of the NCF4 gene were cloned into the pSPL3 vector (Invitrogen, CA, USA). The clones were transformed into Trans5a cells, plated on agar containing 100 mg/ml ampicillin, and incubated overnight at 37°C. Positive colonies were cultured overnight in lysogeny broth at 37°C. Plasmids were isolated with the Endo-free Plasmid Mini Kit II (Omega, USA). The constructs were directly sequenced to verify the presence of the correct sequences.
5
Validating Splice Site Variants
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To ascertain the consequences of novel splice site variants found in our OCA families, the wild and mutant exons along with flanking intronic region (200 bp) were PCR amplified, cloned in pSPL3 vector (Invitrogen, Carlsbad, CA) and sequence verified as described49 (link). Purified cloned constructs were transfected into COS7 cells using PEI (Polyethylenimine). After 48 hours of transfection, RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) and single stranded cDNA (Clontech, Mountain View, CA) was synthesized. Primary PCR amplification of cDNA was performed using SD6 and SA2 vector primers and amplified products were cloned in TA-cloning vector (Invitrogen). At least ten bacterial clones for each construct were Sanger sequenced.
6
Investigating AIFM1 Splice Variants
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The PCR fragments, with or without the AIFM1 c.1265 G > A variant, were constructed into a pSPL3 vector (Invitrogen, California, USA). RNA was extracted from 293 T cells after transfection with each of the two plasmids. RT-PCR was performed to detect splicing variants. The related primers are listed in Table S3 .
7
Mini-Gene Assay for PHEX Mutation
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To examine the predicted results, a mini-gene assay was carried out. We designed a pair of primers (Table 1, PHEX-et_18) covering exon 18 and exonintron boundaries (943 bp). PCR products and pSPL3 vector (Invitrogen Corporation, Carlsbad, CA, USA) were double digested by EcoRI and XhoI followed by a ligation reaction. After transformation into competent cell DH5α, plasmid DNA was prepared by the NucleoBond Xtra Midi Kit (MN, Neumann-Neander-Straße, Germany) for transfection. African green monkey cell line COS7 was cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in 5% CO 2 at 37 °C. We transfected COS7 with empty pSPL3 vector as well as normal/mutant recombinant plasmids using PolyJet reagent (SignaGen Laboratories, Gaithersburg, MD, USA), respectively. After 24 h, cells were harvested and total RNA was extracted. Then reverse transcriptase-PCR was performed using primers SD6 and SA2 (Table 1) followed by agarose gel electrophoresis and sequencing.
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