In Vivo Gene Silencing by Virally Delivered MicroRNA - Viral Vector Approaches in Neurobiology and Brain Diseases - page 252

lentivirally delivered miRNA was suffi cient to induce strong behav-
ioral effect in infected animals. The purpose of these experiments
was to determine whether reduced availability of GRK6 in the
dopamine-depleted striatum of the hemiparkinsonian animals
would affect the frequency of L-DOPA-induced rotations and that
of L-DOPA-induced abnormal involuntary movements (AIMs).
Both rotations and AIMs are considered animal models of
L-DOPA-induced dyskinesia, a frequent and debilitating side effect
of L-DOPA therapy in Parkinson's disease. The results show that
the reduction in the GRK6 concentration caused by miRNA deliv-
ered into the dopamine-depleted striatum increased the rotation
frequency as well as frequency of AIMs [ 68 ], suggesting a role for
GRK6 in L-DOPA-induced dyskinesia.

5 Troubleshooting

The practical recommendations listed below should help the
researchers to successfully use lentivirally delivered miRNA for
gene silencing in the brain of living animals. The main problem
with the miRNA-mediated knockdown experiment is, of course,
failure to achieve measurable knockdown in the brain area of inter-
est in vivo. There are three main reasons why the experiment fails
(1) wrong miRNA, (2) bad viral preparation, and (3) poor virus
injection technique. Only the fi rst problem is specifi c for the
miRNA experiment, whereas the other two are shared with all
studies involving lentivirus-mediated in vivo gene transfer.

1. Selecting an active miRNA sequence is the fi rst critical step in
ensuring success of the miRNA knockdown experiment. In
vitro testing procedure described above is a very helpful and
relatively painless way to select the sequences with potentially
good activity. It is important to conduct the testing in the
right cell type using reasonably good antibody to the protein
of interest to ensure suffi cient sensitivity.
2. If in vitro testing demonstrated good activity of the miRNA
clone but no knockdown is detected in the brain tissue in vivo,
then poor quality of the viral preparation and resulting low
infection effi cacy is the most likely culprit. It is important to
remember that low infection effi cacy in the brain could result
from other causes, so we should consider the quality of the
virus fi rst and evaluate it independently. Since the amount of
the viral particles produced in each preparation will vary, the
titer of the virus in each batch should be determined. There
are many ways to do that (see for example [ 71 ]). We found
that the level of expression of EGFP (expressed cocistronically
with the miRNA) in COS7 cell infected with standard dilution
of the miRNA lentivirus serves well to evaluate the quality of