Differential RNA interference: replacement of endogenous with recombinant low density lipoprotein receptor-related protein (LRP).
The interpretation of experiments involving the overexpression of a recombinant cDNA is often hampered by the interference of mRNA expression from the endogenous gene locus. Unless cell lines from naturally occurring mutations or knockout mice are available, difficult and time-consuming gene targeting techniques are required to inhibit endogenous gene expression. Using a method we refer to as "differential RNA interference" we demonstrate that RNA interference can be used to selectively suppress endogenous gene expression without affecting the expression of a co-transfected recombinant version of the same protein. Functional analyses of recombinant low density lipoprotein receptor-related protein (LRP) to study its involvement in lipid metabolism have been shown to be extremely difficult due to its large cDNA and the unavailability of suitable LRP-deficient cell lines. We constructed an expression vector containing the full-length coding sequence of human LRP fused to EGFP and a vector expressing small hairpin RNA directed against the 3'-untranslated region of the wild-type human LRP mRNA (LRP-shRNA). When overexpressed, EGFP-tagged LRP colocalizes with endogenous LRP and stimulates the uptake of LRP ligands. Overexpression of LRP-shRNA vectors significantly inhibits LRP expression, as judged by quantitative RT-PCR, Western blot and immunofluorescence analysis, and it dramatically decreases receptor-associated protein (RAP) uptake. Finally, co-transfection of EGFP-LRP and LRP-shRNA vectors demonstrates selective inhibition of endogenous LRP expression without affecting simultaneous expression of recombinant LRP protein. Thus, utilization of "differential RNA interference" provides a new experimental approach to selectively study the function of any recombinant protein in any given cell line without interference of endogenous protein expression.
Laatsch
Alexander
A
Institut für Molekulare Zellbiologie, Zentrum für Experimentelle Medizin, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany. laatsch@uke.uni-hamburg.de
Ragozin
Sergei
S
Grewal
Thomas
T
Beisiegel
Ulrike
U
Joerg
Heeren
H
D002460:Cell Line; D015536:Down-Regulation / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000032:analysis; D006801:Humans; D007120:Immunochemistry; D026502:LDL-Receptor Related Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D018613:Microscopy, Confocal; D034622:RNA Interference; D012333:RNA, Messenger / Q000032:analysis; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D026502:LDL-Receptor Related Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins