Minigene

A minigene is a minimal gene fragment that includes an exon and the control regions necessary for the gene to express itself in the same way as a wild type gene fragment. This is a minigene in its most basic sense. More complex minigenes can be constructed containing multiple exons and intron(s). Minigenes provide a valuable tool for researchers evaluating splicing patterns both in vivo and in vitro biochemically assessed experiments. Specifically, minigenes are used as splice reporter vectors (also called exon-trapping vectors) and act as a probe to determine which factors are important in splicing outcomes. They can be constructed to test the way both cis-regulatory elements (RNA effects) and trans-regulatory elements (associated proteins/splicing factors) affect gene expression.

Minigenes were first described as the somatic assembly of DNA segments and consisted of DNA regions known to encode the protein and the flanking regions required to express the protein. The term was first used in a paper in 1977 to describe the cloning of two minigenes that were designed to express a peptide.

RNA splicing was discovered in the late 1970s through the study of adenoviruses that invade mammals and replicate inside them. Researchers identified RNA molecules that contained sequences from noncontiguous parts of the virus’s genome. This discovery led to the conclusion that regulatory mechanisms existed which affected mature RNA and the genes it expresses. Using minigenes as a splice reporting vector to explore the effects of RNA splicing regulation naturally followed and remains the major use of minigenes to date.

In order to provide a good minigene model, the gene fragment should have all of the necessary elements to ensure it exhibits the same alternative splicing (AS) patterns as the wild type gene, i.e., the length of the fragment must include all upstream and downstream sequences which can affect its splicing. Therefore, most minigene designs begin with a thorough in silico analysis of the requirements of the experiment before any "wet" lab work is conducted. With the advent of Bioinformatics and widespread use of computers, several good programs now exist for the identification of cis-acting control regions that affect the splicing outcomes of a gene and advanced programs can even consider splicing outcomes in various tissue types. Differences in minigenes are usually reflected in the final size of the fragment, which is in turn a reflection of the complexity of the minigene itself. The number of foreign DNA elements (exon and introns) inserted into the constitutive exons and introns of a given fragment varies with the type of experiment and the information being sought. A typical experiment might involve wild type minigenes which are expected to express genes normally in a comparison run against genetically engineered allelic variations which replace the wild-type gene and have been cloned into the same flanking sequences as the original fragment. These types of experiments help to determine the effect of various mutations on pre-mRNA splicing.

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