RNA interference of Xenopus NMDAR NR1 in vitro and in vivo.
Here, we present a short interfering RNA (siRNA) application that reduces the expression of NR1 the obligate subunit of the NMDA receptor (NMDAR) and virtually eliminates NMDAR function in a small subset of neurons within otherwise normally developing Xenopus laevis tadpoles. We designed two plasmids each containing a CMV promoter driving a dsRed "reporter" cDNA and DNA coding for one short hairpin RNA (shRNA) under the control of the U6 promoter. The shRNA was cleaved to produce a siRNA against NR1 transcript (iNR1). NR1 transcript and protein, differentiation and survival of NR1 knockdown neurons, were assayed in vivo. iNR1 effects on NMDAR function used Xenopus tectal neuron cultures, Ca2+ imaging and patch-clamp electrophysiology. NR1 transcript, protein and NMDAR function was significantly reduced or eliminated in iNR1-expressing neurons. Protein, transcript and function for the closely related AMPA receptor (AMPAR) subunit GluR1 was unperturbed. In vivo imaging of small groups of iNR1 neurons at two different ages revealed that siRNA knockdown of the NMDAR does not affect differentiation or survival of young neurons. Thus, with siRNA, molecular hypotheses about synaptic mechanisms of circuit formation can be rapidly tested in intact developing vertebrates using normal neurons in the same animals as controls.
Miskevich
Frank
F
McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Frank_Miskevich@tamu-commerce.edu
Doench
John G
JG
Townsend
Matthew T
MT
Sharp
Phillip A
PA
Constantine-Paton
Martha
M
D000818:Animals; D030801:Animals, Genetically Modified; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D004594:Electrophysiology; D004625:Embryo, Nonmammalian; D007814:Larva; D009474:Neurons / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010957:Plasmids / Q000235:genetics; D034622:RNA Interference / Q000502:physiology*; D018091:Receptors, AMPA / Q000235:genetics / Q000502:physiology; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013489:Suppression, Genetic; D014162:Transfection; D014982:Xenopus laevis
C409466:NR1 NMDA receptor; D018091:Receptors, AMPA; D016194:Receptors, N-Methyl-D-Aspartate; D002118:Calcium