Principles and Practical Considerations for the Analysis of Disease-Associated Alternative Splicing Events Using the Gateway Cloning-Based Minigene Vectors pDESTsplice and pSpliceExpress

Abstract

Splicing is an important RNA processing step. Genetic variations can alter the splicing process and thereby contribute to the development of various diseases. Alterations of the splicing pattern can be examined by gene expression analyses, by computational tools for predicting the effects of genetic variants on splicing, and by splicing reporter minigene assays for studying alternative splicing events under defined conditions. The minigene assay is based on transient transfection of cells with a vector containing a genomic region of interest cloned between two constitutive exons. Cloning can be accomplished by the use of restriction enzymes or by site-specific recombination using Gateway cloning. The vectors pDESTsplice and pSpliceExpress represent two minigene systems based on Gateway cloning, which are available through the Addgene plasmid repository. In this review, we describe the features of these two splicing reporter minigene systems. Moreover, we provide an overview of studies in which determinants of alternative splicing were investigated by using pDESTsplice or pSpliceExpress. The studies were reviewed with regard to the investigated splicing regulatory events and the experimental strategy to construct and perform a splicing reporter minigene assay. We further elaborate on how analyses on the regulation of RNA splicing offer promising prospects for gaining important insights into disease mechanisms.

Abnormal Pre-mRNA Splicing in Exonic Fabry Disease-Causing GLA Mutations

“…pDESTsplice was used for the generation of the reporter constructs. To this end, either the wild-type or mutant GLA exon and adjacent introns were inserted as previously suggested [ 20 ] ( Figure 1 b). After transfection of HEK293H cells, total RNA was isolated, cDNA was synthesized and splicing analysis of the minigene was carried out using PCR.…”

Genetic risk variants for multiple sclerosis are linked to differences in alternative pre-mRNA splicing

“…Thus, we studied known splice isoforms and may have missed novel splicing patterns, which can potentially be identified by using RNA sequencing ( 102 , 103 ). Fifth, as we have previously described ( 46 ), there are issues regarding the use of the minigene assay system, such as a possible interference by the Gateway cloning attachment sites, an insufficient amount of an important splicing factor in the used cell line, or the fact that only a small and specific part of the gene is examined. The latter may lead to the misinterpretation of ASEs as the splicing of exons can depend on the correct splicing of other exons of the gene that are not included in the minigene construct.…”

“…While during exon skipping , an exon is excised and not inserted into the mRNA, during intron retention , an intron is not removed and remains in the mRNA molecule. The use of different splice sites can also result in mutually exclusive exons , where only one of two possible exons occurs in the mRNA, or in exons with different lengths due to the use of different acceptor or donor splice sites ( 46 ). In addition to the physiological role of alternative splice sites, genetic variants, such as SNPs, can alter the splicing pattern and thereby contribute to the risk of developing diseases ( 47 ).…”

New Variants of the Cytochrome P450 2R1 (CYP2R1) Gene in Individuals with Severe Vitamin D-Activating Enzyme 25(OH)D Deficiency

“…In the future, we plan to expand this analysis further by performing studies on CYP2R1 and other CYP family enzymes expression and its vitamin D hydroxylation activity in human wild-type and mutant constructs, with a wider patient cohort [ 12 , 17 , 45 ]. The effect of the detected intron variant on splicing will be tested using splicing reporter minigene assay by transient cell lines transfection with a minigene construct [ 60 , 61 ].…”