In Vivo Gene Silencing by Virally Delivered MicroRNA - Viral Vector Approaches in Neurobiology and Brain Diseases - page 235

Chapter 13

In Vivo Gene Silencing by Virally Delivered MicroRNA

Eugenia V. Gurevich , Mohamed R. Ahmed , and Yonatan Carl

Abstract

Loss of function is a standard approach to elucidate the function of a specifi c protein. Among these mul-
tiple strategies for silencing genes in living animals, genetic knockout in mice have been so far most fre-
quently used. However, short hairpin RNAs (shRNAs) and microRNAs (miRNAs) delivered into the brain
by viruses can achieve region-specifi c gene knockdown in any species at much lower cost and with shorter
turnaround time. Recent advances in understanding of the endogenous miRNA function enabled the
design of miRNAs as well as miRNA-adapted shRNA that effi ciently enter the miRNA-processing pathway
and mediate the gene silencing. Predesigned and premade miRNA for many rodent and human genes are
now available commercially. Lentiviral vectors designed to express miRNA along with a fl uorescent marker
are also widely available. Here, we describe the use of virally delivered miRNAs for gene knockdown in
living animals. The technique involves multiple procedures starting from the selection of appropriate
miRNA sequences, then preparation of the lentiviral vector, production of the infections lentivirus suitable
for the in vivo delivery, injection of the virus into the brain, and the testing of the animals for the measure
of interest such as behavior, and, fi nally, post hoc determination of the infection effi ciency and the degree
of the in vivo knockdown in each animal.
The virally delivered miRNA knockdown is powerful enough to achieve physiologically relevant protein
knockdown in the brain of living animals. Furthermore, the knockdown procedure is fl exible enough to be
adapted to requirements of almost any in vivo experiment, and, thus, has a large yet unrealized potential.

Key words MicroRNA, Short hairpin RNA, Lentivirus, Viral vector, In vivo gene knockdown, Gene
silencing, Brain injection, Virus injection

1 Background and Historical Overview

The ability to selectively decrease the expression of proteins, or
loss-of-function approach, is very useful for the study of the func-
tions of the proteins of interest. Gene knockdown, or the reduc-
tion of the concentration of the protein of interest, has become
standard element in the experimental repertoire in molecular and
cell biology. The discovery of the mechanisms of RNA interference
(RNAi) emerged from studies of gene silencing fi rst with antisense
nucleotides and later with double-stranded RNAs (dsRNA) ([ 1 , 2 ],
see also [ 3 ] for review). It was later discovered that dsRNAs are
cleaved by the RNAse III enzyme Dicer that generates shorter