UNIT 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Cell Biology
How to Cite
Rooke, N. and Underwood, J. 2001. In Vitro RNA Splicing in Mammalian Cell Extracts. Current Protocols in Cell Biology. 18:11.17:11.17.1–11.17.20.
- Published Online: 1 MAY 2001
- Published Print: OCT 1998
Almost every eukaryotic pre-mRNA generated by RNA polymerase II transcription requires the removal of introns to create mRNA. The correct splicing of constitutive exons is thus critical for normal protein expression and function. Moreover, the removal of many introns by the spliceosome is controlled in a tissue-specific or developmentally specific manner. In order to study RNA splicing at a biochemical level, it is necessary to employ an in vitro, or cell-free, system. Cell-free splicing systems require two main components: (1) an extract made from mammalian cell nuclei and (2) the introns and exons of the eukaryotic gene of interest. This minigene construct allows the synthesis of sufficient quantities of pre-mRNA substrates in vitro, which are then incubated in the nuclear extract and analyzed for splicing. Nuclear extracts, first developed for studying transcription in vitro, are modified for splicing. This unit describes how to set up an in vitro splicing reaction using a mammalian nuclear extract derived from either cell line or tissue, and how to analyze the splicing reaction products.