Minigene

A minigene is a simplified gene construct used to study RNA splicing and gene expression in a controlled cellular environment. It typically comprises a small genomic fragment of interest—usually one or a few exons with surrounding intronic sequences—placed within an expression vector under the control of a promoter. The transcript produced by the host cell is spliced, and the resulting RNA products are analyzed by techniques such as RT-PCR, sequencing, or reporter readouts to assess splicing outcomes. Many minigenes also include a reporter gene or selectable marker to facilitate quantification of expression.

Construction and use: A minigene is designed to isolate the splicing event under study from other regulatory elements present in the full gene. Common design elements include a test exon flanked by introns, a constitutive or tissue-specific promoter, and a polyadenylation signal. The construct is introduced into cultured cells or model organisms, and splicing patterns are assessed in the appropriate cellular context. Minigenes can be used to test the effects of specific sequence variants on splice site selection, enhancer or silencer elements, and to compare splicing across cell types.

Applications and limitations: Minigene assays are used to characterize splicing defects associated with genetic variants, to study regulatory sequences governing exon inclusion, and to screen for splicing-modulating compounds. They provide a rapid and cost-effective system but may not fully recapitulate endogenous regulation because of the simplified genomic context, lack of chromatin structure, and artificial promoter effects. Results should be interpreted with these caveats and validated in more native models when possible.