Table 2.
Experimental setup for the minigene assays applied in the 25 studies.
| Reference | Genes | Exons | Genetic Variants | Source (Size) | Variant Creation | Donor Vector | Cloning | Construct Verification | Cells | RNA Iso. | Detection Procedures |
|---|---|---|---|---|---|---|---|---|---|---|---|
| Abdulhay et al., 2019 [55] | GATA1 | Exon 5–6 | chrX:48652176C>T (hg19) | DNA (1335 bp *) | Control & patient | / | RE | N/A | HEK293T | 48 h | RT-PCR, Gel electrophoresis, Sequencing (PCR product) |
| Alaa el Din et al., 2015 [46] | ACVRL1 | Exon 6, Exon 7, Exon 9 |
c.733A>G, c.1249A>T, c.1048+5G>A |
DNA (≈500–700 bp) | N/A | / | Gateway | RE digestion, Sequencing (plasmid) | HeLa | 48 h | RT-PCR, Gel electrophoresis, Sequencing (PCR product) |
| Bartosovic et al., 2017 [49] | BRD8 | Exon 20–21 | / | Gene synthesis (1202 bp *) | Site-directed mutagenesis | N/A | Gibson Assembly | N/A | HEK293T | 24 h | RT-PCR, Gel electrophoresis |
| Beaman et al., 2019 [57] |
CHRM3 | Exon 7 * | c.352G>A | DNA (840 bp) | Control & patient | / | NEBuilder® | Sequencing (plasmid) | HEK293 | 20 h | RT-PCR, Gel electrophoresis, Sequencing (PCR product) |
| Cao et al., 2020 [62] |
SS18L1, CAP1, IFT122 | Exon 2–3 (SS18L1) *, Exon 2 (CAP1) *, Exon 8–9 (IFT122) * |
pMEIs | DNA (≈2800–5500 bp *) | N/A | / | Gateway | Sequencing (plasmid) | HEK293T | 24 h | RT-PCR, Gel electrophoresis |
| Carvill et al., 2018 [50] |
SCN1A | Exon 20–21 | chr2:166864064G>A, chr2:166864057_166864061del, chr2:166863778C>G, chr2:166863774C>T, chr2:166863726G>A (hg19) | DNA (≈7500 bp) | Site-directed mutagenesis | pDONR221 | Gateway | Sequencing (plasmid) | K562, A549 | 24 h | RT-qPCR |
| Chase et al., 2020 [60] |
EZH2 | Exon 8 | Y244D, E249K, L252V, A255T, R288Q, H297R, R298L | BAC-derived PCR fragment (1543 bp *) |
Site-directed mutagenesis | / | Gateway | N/A | HEK293F, HeLa | 48 h | RT-PCR, Gel electrophoresis |
| Dupont et al., 2019 [58] |
IFT52 | Exon 8 | c.695–699delinsCA | DNA (464 bp *) | Control & patient | / | Gateway | N/A | HEK293T | 48 h | RT-PCR, Gel electrophoresis |
| Ellingford et al., 2019 [59] | ABCA4, GUCY2D, PDE6B, MERTK, SCN2A, ABHD12, CRYBA1, DNAH11, CFTR, RPGR, MYBPC3, TRPM1 |
N/A | NM_000350.2:c.5584+6T>C, NM_000180.3:c.3043+5G>A, NM_000283.3:c.2130-15G>A, NM_006343.2:c.2486+6T>A, NM_001040142.1:c.2919+3A>G, NM_015600.4:c.867+5G>A, NM_005208.4:c.213C>T, NM_001277115.1:c.6547-963G>A, NM_000492.3:c.3874-4522A>G, NM_001034853.1:c.247G>T, NM_001034853.1:c.1754-3G>C, NM_000256.3:c.1224-21A>G, NM_002420.5:c.899+29G>A | DNA (N/A) | Control & patient | / | NEBuilder® | Sequencing (plasmid) | HEK293 | 48 h | RT-PCR, Gel electrophoresis, Sequencing (PCR product) |
| Reference | Genes | Exons | Genetic Variants | Source (Size) | Variant Creation | Donor Vector | Cloning | Construct Verification | Cells | RNA Iso. | Detection Procedures |
| Kishore et al., 2010 [41] |
DPM2, TAF1, RALGPS1, PBRM1, CRHR1 |
N/A | / | BAC-derived PCR fragments (N/A) |
/ | / | Gateway | RE digestion, Sequencing (plasmid) | Neuro2A | N/A | RT-PCR, Gel electrophoresis |
| Knapp et al., 2020 [63] |
DONSON | Exon 3–5 | c.607-36G>A | DNA (3130bp *) | Control & patient | / | Gateway | Sequencing (plasmid) |
HeLa | 24 h | RT-PCR, Gel electrophoresis, Sequencing (PCR product) |
| Legendre et al., 2018 [51] | CHD7 | Exon 26 | rs398124321, rs1131690787, rs794727423, rs199981784 | DNA (566bp) | Site-directed mutagenesis | / | Gateway | Sequencing (plasmid) |
HeLa | 48 h | RT-PCR, Fluorescent capillary electrophoresis, Nested lariat RT-PCR, Sequencing (PCR product) |
| Listerman et al., 2013 [44] | TERT | Exon 5–9 | / | DNA from HeLa (N/A) | / | / | Gateway | RE digestion, Sequencing (plasmid) | HEK293T | 48 h | RT-qPCR |
| Mattison et al., 2018 [53] |
SLC6A1 | Exon 8–10 | c.850-2A>G | DNA (1450bp) | Site-directed mutagenesis | pENTR/D- TOPO |
Gateway | RE digestion, Sequencing (plasmid) | HEK293 | 24 h | RT-PCR, Gel electrophoresis, Sequencing (PCR product) |
| Mutai et al., 2020 [64] |
SLC12A2 | Exon 21–22 | c.2930-2A>G | DNA (2507bp) | Control & patient | / | Gateway | Sequencing (plasmid) | HEK293T | 48 h | RT-PCR, Gel electrophoresis, Sequencing (PCR product) |
| Payer et al., 2019 [54] |
NUP160, CCDC110, BPIFC, SLC2A9, CD58 | Exon 33 (NUP160), Exon 5 (CCDC110), Exon 10–11 (BPIFC), Exon 2 (SLC2A9), Exon 3 (CD58) | AluYh3a3 (ALU_umary_ALU_8566), AluY (ALU_umary_ALU_4001), AluYa5 (ALU_umary_ALU_12481), AluYi6 (RIP-041), AluY (DEL_pindel_1315) |
DNA, Gene synthesis (≈1000–2400bp *) |
Control & patient, Gene synthesis |
/ | Gateway, Gibson Assembly | Sequencing (plasmid) | HEK293T | 24 h | RT-PCR, Gel electrophoresis |
| Rittore et al., 2014 [45] |
TNFRSF1A | Exon 1–4, Exon 1–2, Exon 2–4 | rs1800692, rs4149570, rs767455 | DNA (≈800–1600bp) | Site-directed mutagenesis | TOPO-TA | RE | N/A | HEK293T, SW480 | N/A | RT-qPCR |
| Scott et al., 2012 [43] |
CFTR | Exon 6, Exon 8, Exon 15, Exon 22 |
rs35033453, rs1800083, rs1800084, rs1800105, rs1800122 | Gene synthesis (≈250–400bp) | Gene synthesis |
TOPO-TA (pCR™8) | Gateway | RE digestion, Sequencing (plasmid) | K562, IB3-1 | 24 h | RT-PCR, Gel electrophoresis |
| Starokadomskyy et al., 2016 [48] |
POLA1 | Exon 13–14 | NC_000023.10:g.24744696A>G | DNA (N/A) | Control & patient | / | Gateway | Sequencing (plasmid) | HEK293 | 48 h | RT-PCR, Gel electrophoresis, Sequencing (PCR product) |
| Reference | Genes | Exons | Genetic Variants | Source (Size) | Variant Creation | Donor Vector | Cloning | Construct Verification | Cells | RNA Iso. | Detection Procedures |
| Sumanasekera et al., 2012 [42] |
DBF4B, MYO18A b, POLB, MAPT, SYK |
Exon 10 (DBF4B) *, Exon 39–41 (MYO18A) *, Exon 1–2 (POLB) *, N/A (SYK, MAPT) |
/ | BAC-derived PCR fragments (≈2200–4400bp *) |
/ | / | Gateway | RE digestion, Sequencing (plasmid) | HEK293 | N/A | RT-PCR, Gel electrophoresis |
| Tang et al., 2020 [65] |
CDH23, SLC9A3R1 | Exon 50 (CDH23), Exon 3 (SLC9A3R1) |
rs56013867, rs41282067 | Gene synthesis (≈150–230 bp) | Gene synthesis |
pENTR/D- TOPO |
Gateway | RE digestion, Sequencing (plasmid) | HEK293 | 24 h | RT-PCR, Gel electrophoresis, Sequencing (PCR product) |
| Thomas et al., 2020 [61] |
EFTUD2 | Exon 5, Exon 9, Exon 10, Exon 13 Exon 15, Exon 16, Exon 18, Exon 19, Exon 20, Exon 23, Exon 25, Exon 26, Exon 27 |
c.428C>T, c.620G>A, c.623A>G, c.670G>A, c.670G>C, c.784C>T c.857A>G, c.1149G>C, c.1306C>G, c.1426T>C, c.1496G>A, c.1732C>T, c.1860G>C, c.1860G>T, c.1910T>G, c.2033C>A, c.2305G>C, c.2332C>T, c.2467G>A, c.2485G>A, c.2566C>T, c.2813G>A | DNA (N/A) | Site-directed mutagenesis | / | Gibson Assembly | Sequencing (plasmid) | HEK293 | 48 h | RT-PCR, Gel electrophoresis, Sequencing (PCR product) |
| Varga et al., 2019 [56] |
EYA4 | Exon 10 | c.804G>C | DNA (N/A) | Patients | / | Gateway | Colony PCR, Sequencing (plasmid) | HeLa | 24 h | RT-PCR, Gel electrophoresis, Sequencing (PCR product) |
| Wang et al., 2018 [52] |
Lrp8 a, Grin1 a |
Exon 19 (Lrp8), Exon 21 (Grin1) |
/ | N/A (≈1000–1400bp *) | Site-directed mutagenesis | / | N/A | N/A | HeLa | 48 h | RT-PCR, Gel electrophoresis |
| Xiao et al., 2016 [47] |
ZNF638 | Exon 2 | / | DNA from HeLa (≈2500bp) | Site-directed mutagenesis | / | Gateway | N/A | HeLa | 48 h | RT-PCR, Gel electrophoresis |
Alternative splicing events of 51 different genes were examined. The Gateway vectors pDESTsplice and pSpliceExpress and the used donor vectors for the Entry clone creation are given in the table. If no information from the study was available, it was marked as N/A in the corresponding field, and if a procedure was not used in the study, a slash was noted (/). In cases where genetic variants were examined, they were recorded in the table. The investigated exons and the source of the genomic sequence of interest, namely, genomic DNA from donors, DNA extracted from cell lines, gene synthesis fragments, or BAC-derived PCR fragments, are recorded. In addition, the fragment size is given. If only one exon was examined, the exact size was noted; however, if several exons have been examined, the range of the approximate insert size is given. In cases where the examined exons or the insert sizes have not been described but could be calculated on the basis of the given primers, the specifications in the table are marked with an asterisk (*). We also recorded how the investigated variants were created, which cloning procedure was applied, and how the minigene constructs were verified. Furthermore, we noted the cell lines, which were transiently transfected with the minigene constructs and checked how long they were incubated before the RNA was isolated. Procedures used to detect the transcribed minigene RNA molecules were RT-PCR followed by gel electrophoresis or fluorescent capillary electrophoresis, nested lariat RT-PCR, RT-qPCR, and (Sanger) sequencing. a Investigations were performed on genes from mice. b According to the current human genome assembly of the UCSC Genome Browser (Dec. 2013, hg38), the investigated splicing event concerns the MYO18A gene and not the TIAF1 gene as annotated in previous genome assemblies. BAC: bacterial artificial chromosome, N/A: not available, RE: restriction enzyme, RNA iso: RNA isolation time point (post-transfection), RT-PCR: reverse transcriptase polymerase chain reaction, RT-qPCR: quantitative reverse transcription polymerase chain reaction.