Unit

UNIT 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts

  1. Nanette Rooke,
  2. Jason Underwood

Published Online: 1 MAY 2001

DOI: 10.1002/0471143030.cb1117s18

Current Protocols in Cell Biology

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.

Author Information

  1. University of California, Los Angeles, Los Angeles, California

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: OCT 1998

Abstract

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.