Gene Therapy for Parkinson’s Disease - Viral Vector Approaches in Neurobiology and Brain Diseases - page 177

parameters should be evaluated in an appropriate model of PD,
such as the 6-hydroxydopamine (6-OHDA) rodent model or
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse or
primate models (for a recent review on animal model for PD
research see Duty and Jenner [ 35 ]) ( see Note 3 ). To easily visualize
the effi ciency and specifi city of the promoter candidates, it is rec-
ommended to clone the individual candidates into an appropriate
viral vector system expressing a reporter gene such as green fl uores-
cent protein (GFP) in the evaluation experiments ( see Note 4 ).
The virus may then be introduced into an appropriate nucleus in
the CNS by stereotaxic injection ( see Notes 5 - 7 ). It is recom-
mended to wait at least 3 weeks after viral injection before perfu-
sion or dissection of the brain to ensure a stable transgene
expression.

The characteristics of the vectors carrying the different promoter
candidates can be analyzed by immunohistochemical procedures
and analysis of the autofl uorescence (if a fl uorescent transgene, such
as GFP or RFP, was used) on cryosectioned brain slices ( see Note
8 ). The cell specifi city may be analyzed by double staining for the
transgene in combination with a neuronal marker, such as NeuN, or
a glial marker, such as GFAP. The colocalization of 200-250 cells is
then determined by microscopy. The obtained specifi city can then
be compared to conventionally used neuron-specifi c promoters
NSE and synapsin (Syn), which generally show a neuronal specifi c-
ity of around 98 %, and glial-specifi c promoter GFAP, which gener-
ally show a glial specifi city of 70 % [ 27 , 36 ]. The effi ciency of the
promoter in vivo may be evaluated by counting the total number of
GFP-positive cells and divide it with the total number of transform-
ing units (TU) injected. The value can then be compared to the
effi ciency of ubiquitous promoters CMV, elongation factor 1 alpha
(EF1

2.3 Postmortem
Analysis

), and CAG (CMV and chicken beta-actin hybrid promoter),
which is generally around 7-18 % [ 27 ]. The level of transgene
expression may be evaluated by measuring the autofl uorescence of
the sections if a fl uorescent transgene is used. Alternatively, cDNA
can be prepared from fresh frozen tissue and the level of transgene
messenger RNA can be estimated by RT-qPCR.

α

2.4 Fine-Tuning of
Transgene Expression
in the Brain Using
miRNA Detargeting

Although many promoters are highly specifi c for one cell type,
there may still be some transgene expression in other cell types.
Transgene expression could potentially be silenced in these
unwanted cell types using miRNA detargeting. The selection of a
particular miRT will be dependent on species, cell type, brain
region, and developmental state. It is known that for an effi cient
gene regulation, the miRNA mediating the regulation will need to
be highly expressed in the cell type/brain area in question (>100
copies/cell) to ensure the messenger mRNA will be effi ciently