In Vivo Analysis of Splicing Assays

The use of reporter minigenes in transfection assays, commonly referred to as in vivo splicing assays, is the most frequently used method to analyze alternative splicing patterns. The splicing reporters can easily be transfected in a large number of cell lines. The use of a splice site can then be assayed by using RT-PCR or qRT-PCR. An in vivo splicing assay can serve as an accurate, rapid, and simple method to reproduce splicing events seen in an intact organism. For example, it can serve to study the effects of the knock-down or the over-expression of any factor on the studied splicing event. For larger screens, adapted reporters may be constructed. Splicing reactions may then be studied without RNA isolations, followed by RT-PCR reactions. Following the construction of stable cell lines, a splicing event can be analyzed on a larger scale, simply by using luciferase or fluorescence assays. In the laboratory, two types of analysis have been developed to study the regulation of one single splicing event that is responsible for the majority of the cases of a rare disease, Hutchinson–Gilford progeria syndrome.