Preparation of Hela Cell Nuclear and Cytosolic S100 Extracts for In Vitro Splicing

Following the initial discovery of split genes in 1977, it took several years before in vitro systems were successfully developed to study the biochemistry of pre-mRNA splicing. The first systems relied on coupling of transcription and splicing in whole-cell extracts and were fairly inefficient, because of the different optima for these two reactions (1 ,2 ). It was later shown that these reactions could be uncoupled (3 ,4 ), but obtaining discrete pre-mRNAs in useful amounts remained an obstacle until in vitro transcription with bacteriophage RNA polymerases (3 ) was adopted for this purpose. Another useful development was a nuclear extract preparation procedure that was initially developed for in vitro transcription studies (5 ). No splicing was detected in this study. However, the same extract preparation procedure, in conjunction with pre-mRNAs transcribed from cloned genes by SP6 RNA polymerase, was used to define optimal conditions for in vitro splicing (6 ). This system results in relatively efficient and accurate splicing, and is now in wide use, with slight variations from laboratory to laboratory. Variations in extract preparation include primarily the use of slightly different buffers and salts for nuclear extraction or dialysis.