Krichevsky AM, Kosik KS. RNAi functions in cultured mammalian neurons. Proc Natl Acad Sci U S A. 2002 Sep 3;99(18):11926-9. PubMed Abstract

REAGENTS/MATERIAL:

Primary rat (Sprague-Dawley) embryonal neuronal cells cultured in Neurobasal medium containing B27 supplement and 0.5mM glutamine.

siRNAs corresponding to pEGFP and dsRed2 reporter genes and to MAP2 or YB-1 mRNAs were synthesized by Xeragon.

transfections performed using Lipofectamine 2000 (Invitrogen).TransMessenger transfection reagent(Qiagen)used with siRNA for endogenous gene targeting.

Antibodies: mouse monoclonal anti-MAP2 (1:200)produced in the laboratory; Rabbit polyclonal anti-YBI (p50) provided by V. Evdokinova of McGill Univ., Montreal, QC,Canada; rabbit polyclonal anti-cortactin (Santa Cruz Biotechnology); Alexa 488 or Alexa 594-conjugated goat anti-mouse secondary ab (Molecular Probes); Alexa Fluor 594 phalloidin (Molecular Probes).

Fluorescent microscopy performed with a confocal laser scanning unit. Pixel activity quantified by Adobe Photoshop software.

FUTURE DIRECTION:
This work seems to demonstrate that the cellular machinery that is necessary for RNAi is functional in post-mitotic neurons. The technique itself may need some refining, but once the technical hurdles are crossed it will be interesting to see what RNAi-mediated gene silencing reveales in cultured neurons.