Nearly a decade ago, now-Nobel laureates Craig Mello and Andrew Fire discovered that they could insert short RNA molecules into worms and shut down specific genes. Today, scientists routinely use this powerful method, termed RNA interference, to study the functions of specific genes in mammalian systems.
In order to conduct these experiments, scientists generally rely on chemical synthesis of RNA molecules, which can be quite costly. A freely accessible article from this month’s release of Cold Spring Harbor Protocols (www.cshprotocols.org) addresses this problem; it describes a cost-effective approach for generating silencing RNAs, called esiRNAs, to efficiently target virtually any gene in mammalian cells.
The protocol (http://www.cshprotocols.org/cgi/content/full/2007/16/pdb.prot4824) describes how to enzymatically generate RNA molecules in vitro, using the cloned gene of interest as a template. The RNA molecules are then randomly cleaved into short fragments, purified, and used in RNA interference experiments.
The procedure was developed by Dr. Frank Buchholz’s group at the Max Planck Institute of Molecular Cell Biology and Genetics (Germany), and can be used to generate large sets of esiRNA libraries to be applied to large-scale studies of gene function (http://www.mpi-cbg.de/esiRNA/).
Also highlighted in Cold Spring Harbor Protocols this month is an article that describes how to culture thymus cells from fetal mice (http://www.cshprotocols.org/cgi/content/full/2007/16/pdb.prot4808). The thymus is the organ where T-cells—a principal component of the immune system in vertebrates—proceed through a strictly coordinated maturation process before being released into the bloodstream. Fetal thymus organ culture is the only system available for studying the complete program of T-cell maturation in vitro, and the protocol will be useful to researchers interested in understanding the intricacies of T-cell maturation. It was authored by Drs. Graham Anderson and Eric J. Jenkinson from the MRC Centre for Immune Regulation at the University of Birmingham (U.K.) (http://www.mrcbcir.bham.ac.uk/research/t-celldevelopment.htm).
Other articles published today include methods for imaging neuronal activity in zebrafish, examining gene expression patterns in fruit flies and frogs, preparing DNA from mammals for genotyping, and identifying protein-protein interactions in virtually any species. For a complete list of articles in the August release of Cold Spring Harbor Protocols, please see http://www.cshprotocols.org/TOCs/toc8_07.dtl
