The development of small-interfering RNA (siRNA)-mediated gene-silencing strategies has made it possible to study the transport of precursors of soluble and membrane proteins into the endoplasmic reticulum (ER) of human cells. In these approaches, a certain target gene is silenced in the cell type of choice, followed by analysis of the effect of this silencing on the biogenesis of a single or set of precursor polypeptide(s) in cell culture or in cell-free assays involving semi-permeabilized cells and in vitro translations systems. These approaches allow for functional analysis of components of the ER-resident protein transport machinery as well as the elucidation of their potential cell-type variations and regulatory mechanisms. The gene-silencing and subsequent plasmid-based complementation carries the additional benefit of facilitating analysis of the consequences of disease-linked mutations in ER transport components.

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http://dx.doi.org/10.1007/978-1-62703-487-6_18DOI Listing

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