Document Detail

Small-scale nuclear extracts for functional assays of gene-expression machineries.
MedLine Citation:
PMID:  22782264     Owner:  NLM     Status:  MEDLINE    
A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.
Eric G Folco; Haixin Lei; Jeanne L Hsu; Robin Reed
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Video-Audio Media     Date:  2012-06-27
Journal Detail:
Title:  Journal of visualized experiments : JoVE     Volume:  -     ISSN:  1940-087X     ISO Abbreviation:  J Vis Exp     Publication Date:  2012  
Date Detail:
Created Date:  2012-07-11     Completed Date:  2012-09-17     Revised Date:  2013-07-12    
Medline Journal Info:
Nlm Unique ID:  101313252     Medline TA:  J Vis Exp     Country:  United States    
Other Details:
Languages:  eng     Pagination:  -     Citation Subset:  IM    
Department of Cell Biology, Harvard Medical School, MA, USA.
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MeSH Terms
Cell Nucleus / chemistry*,  genetics*
Epoxy Compounds / pharmacology
Gene Expression Regulation*
HeLa Cells
Macrolides / pharmacology
RNA Polymerase II / chemistry,  genetics
Grant Support
Reg. No./Substance:
0/E 7107; 0/Epoxy Compounds; 0/Macrolides; EC 2.7.7.-/RNA Polymerase II

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