Targeting the kinesin Eg5 to monitor siRNA transfection in mammalian cells.
RNA interference, the inhibition of gene expression by double-stranded RNA, provides a powerful tool for functional studies once the sequence of a gene is known. In most mammalian cells, only short molecules can be used because long ones induce the interferon pathway. With the identification of a proper target sequence, the penetration of the oligonucleotides constitutes the most serious limitation in the application of this technique. Here we show that a small interfering RNA (siRNA) targeting the mRNA of the kinesin Eg5 induces a rapid mitotic arrest and provides a convenient assay for the optimization of siRNA transfection. Thus, dose responses can be established for different transfection techniques, highlighting the great differences in response to transfection techniques of various cell types. We report that the calcium phosphate precipitation technique can be an efficient and cost-effective alternative to Oligofectamine in some adherent cells, while electroporation can be efficient for some cells growing in suspension such as hematopoietic cells and some adherent cells. Significantly, the optimal parameters for the electroporation of siRNA differ from those for plasmids, allowing the use of milder conditions that induce less cell toxicity. In summary, a single siRNA leading to an easily assayed phenotype can be used to monitor the transfection of siRNA into any type of proliferating cells of both human and murine origin.
Weil
D
D
CNRS UPR1983, Institut André Lwoff, 7 rue Guy Moquet, 94801 Villejuif, France.
Garçon
L
L
Harper
M
M
Duménil
D
D
Dautry
F
F
Kress
M
M
D002130:Calcium Phosphates; D002448:Cell Adhesion; D002463:Cell Membrane Permeability; D011232:Chemical Precipitation; D003362:Cost-Benefit Analysis; D004337:Drug Carriers; D018274:Electroporation; D018390:Gene Targeting / Q000191:economics / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D020014:K562 Cells; D016547:Kinesins / Q000235:genetics*; D007947:Leukemia, Megakaryoblastic, Acute / Q000473:pathology; D008938:Mitosis / Q000187:drug effects; D034622:RNA Interference; D012333:RNA, Messenger / Q000037:antagonists & inhibitors; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology*; D014162:Transfection / Q000191:economics / Q000379:methods*; D014407:Tumor Cells, Cultured; D029867:Xenopus Proteins / Q000235:genetics*
D002130:Calcium Phosphates; D004337:Drug Carriers; C088335:KIF11 protein, Xenopus; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D029867:Xenopus Proteins; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; D016547:Kinesins; C485829:calcium phosphate, dibasic, anhydrous