Local Knockdown of Genes in the Brain Using Small Interfering RNA: A Phenotypic Comparison with Knockout Animals

doi:10.1016/j.biopsych.2006.03.020

Biological Psychiatry

Volume 61, Issue 1, 1 January 2007, Pages 65-69

https://doi.org/10.1016/j.biopsych.2006.03.020 Get rights and content

Background

Recent reports have suggested effectiveness of RNA interference (RNAi) for the analysis of gene functions in the brain. This study sought to determine the efficiency of local small interfering RNA (siRNA) injections, comparing this approach with animals generated through classical gene targeting.

Methods

Small interfering RNA against dopamine transporter (DAT) (35 μg/14 days) or tyrosine hydroxylase (TH) (15 μg/3 days) was injected into the ventral tegmental/substantia nigra areas of the brain of adult wildtype or DAT-knockout mice, respectively.

Results

Local injections of siRNA resulted in a 35% to 40% reduction of DAT and TH protein levels in the striatum, respectively. Despite negligible effect of DAT knockdown on novelty-induced locomotion, the locomotor response of DAT siRNA treated animals to amphetamine was blunted similar to what is observed in the DAT heterozygote animals. Since incomplete reduction of TH levels in normal mice does not produce behavioral effects, TH siRNA experiments were carried out in DAT-knockout animals that show increased dependence on newly synthesized dopamine. Knockdown of TH in these animals resulted in reduced basal locomotion.

Conclusions

Local injection of siRNA in the brain reduced gene expression by 40% to 50%, suggesting that siRNA-mediated knockdown of genes in the brain can be a complementary tool to classical transgenesis for the analysis of gene functions.

Section snippets

Animals

Male C57BL/6J mice were used for experiments. Mice were 3 to 5 months old at the time of the experiments. All mice were group housed (4 to 5 per cage) in a humidity- and temperature-controlled room with 12-hour/12-hour light/dark cycle (lights on at 8:00 am). All animal experiments were performed during the light cycle. Mice were provided food and water ad libitum. Dopamine transporter knockout mice were generated by disruption of the DAT gene through homologous recombination (Giros et al 1996

Knockdown of the Dopamine Transporter in Mice by siRNA Injections

As a first step to evaluate the use of nude siRNA in vivo, knockdown of DAT was attempted by performing (ICV) injections of DAT siRNA. Since it has been reported that the half-life of DAT in the brain of rodents is between 5 to 7 days (Fleckenstein et al 1996, Kimmel et al 2000), mice were injected with a mix of 5 μg of siRNA and .5 μg of the lipid carrier DOTAP every other day for a period of 14 days (total seven injections). This injection regimen resulted in a downward trend (∼15%) of DAT

Discussion

The use of siRNA has proven to be an interesting emerging tool to disrupt gene function in mammalian cells in vitro. The ability to achieve similar knockdowns in the brain is desirable and would allow for rapid annotation and functional analysis of genes potentially implicated in neurological disorders by circumventing the need to generate transgenic animals. In this study, local injections of siRNA were performed to achieve knockdowns of DAT and TH, and the phenotypic behavior of DAT knockdown

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Original articleLocal Knockdown of Genes in the Brain Using Small Interfering RNA: A Phenotypic Comparison with Knockout Animals

Background

Methods

Results

Conclusions

Section snippets

Animals

Knockdown of the Dopamine Transporter in Mice by siRNA Injections

Discussion

J Neurosci Methods

FEBS Lett

Neuropharmacology

Neuron

Cell

Late onset of motor neurons in mice overexpressing wild-type peripherin

J Cell Biol

Lithium antagonizes dopamine-dependent behaviors mediated by an AKT/glycogen synthase kinase 3 signaling cascade

Proc Natl Acad Sci U S A

Biosynthesis of tyrosine hydroxylase in the rat adrenal medulla after exposure to cold

Proc Natl Acad Sci U S A

Turnover rate of tyrosine hydroxylase during trans-synaptic induction

Mol Pharmacol

Recovery of dopamine transporter binding and function after intrastriatal administration of the irreversible inhibitor RTI-76 [3 beta-(3p-chlorophenyl) tropan-2 beta-carboxylic acid p-isothiocyanatophenylethyl ester hydrochloride]

J Pharmacol Exp Ther

Monoamine transporters: From genes to behavior

Annu Rev Pharmacol Toxicol

Role of serotonin in the paradoxical calming effect of psychostimulants on hyperactivity

Science

Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter

Nature

RNA interference

Nature

Original article
Local Knockdown of Genes in the Brain Using Small Interfering RNA: A Phenotypic Comparison with Knockout Animals