Preparation of Efficient Splicing Extracts From Whole Cells, Nuclei, and Cytoplasmic Fractions
Pre-mRNA (messenger RNA) splicing is an essential step for gene expression in higher eukaryotes. Splicing reactions have been well studied in vitro using extracts prepared from cultured cells. We describe protocols for the preparation of splicing-competent extracts from whole cells, nuclei, and cytoplasmic fractions. The nuclear and whole-cell extracts are fully active in splicing, while S100 extracts are able to support splicing only when SR (Serine/Arginine-rich) proteins are supplied. The simple method described here to prepare splicing active extracts from whole cells is particularly useful in studying pre-mRNA splicing in many different cell types.
Key Words:Nuclear extracts S100 extracts sonication splicing whole-cell extracts.
- 20.Sakamoto, H., Ohno, M., Yasuda, K., Mizumoto, K., and Shimura, Y. (1987) In vitro splicing of a chicken delta-crystallin pre-mRNA in a mammalian nuclear extract. J. Biochem. (Tokyo) 102, 1289–1301.Google Scholar