Rab1b silencing using small interfering RNA for analysis of disease-specific function.
Rab1 GTPase is a critical component required for endoplasmic reticulum (ER)-to-Golgi as well as intra-Golgi trafficking. It is required for the proper recruitment of tethering factors to mediate vesicle docking and subsequent fusion to the target membrane compartment. Much is known about the role of Rab1 in ER-to-Golgi trafficking through overexpression of dominant negative mutation that inhibit GTP binding or GTPase activity of the protein, as well as through the use of antibodies to inhibit endogenous protein activity. These techniques have allowed for the establishment of a central role for Rab1 in trafficking in the early secretory pathway; however, the use of these techniques is limited. The introduction of antibodies relies on permeabilization of the cell membrane for their introduction. The use of dominant negative mutations relies on the mutation overwhelming the endogenous protein activity without removing it from the cell. The advent of siRNA to silence genes of interest provides a means to overcome this limitation. Here we describe optimal conditions for the efficient silencing of Rab1b using siRNA to analyze its role in disease.
Hutt
Darren M
DM
Department of Cell Biology, The Scripps Research Institute, La Jolla, California, USA.
Balch
William E
WE
D002478:Cells, Cultured; D003550:Cystic Fibrosis / Q000503:physiopathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D020868:Gene Silencing; D006801:Humans; D034741:RNA, Small Interfering / Q000378:metabolism*; D020545:Respiratory Mucosa / Q000166:cytology; D014162:Transfection / Q000379:methods; D020761:rab1 GTP-Binding Proteins / Q000235:genetics*
D034741:RNA, Small Interfering; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C529587:Rab1B protein, human; D020761:rab1 GTP-Binding Proteins