Biology Reference
In-Depth Information
2.8 Histological
Analysis
1. Cryostat.
2. Round cryostat chuck 1-1/2
″
(40 mm).
3. Sectioning Blades.
4. Trimming Blades.
5. Glass slides.
6. Dissecting microscope.
7. 4
,6-Diamidino-2-phenylindole (DAPI).
8. Rectangular coverslips (24 × 60).
9. Transparent nail-polish.
10. Fluorescence microscope.
′
3
Methods
The methods described in this chapter include: Subheading
3.1
the generation of plasmids for AAV vector production including
expression cassettes encoding for EPO and the EPO derivative
S100E. Subheading
3.2
the expression analysis of the EPO
and S100E proteins in vitro; Subheading
3.3
the subretinal and
intramuscular injections of AAV vectors encoding EPO and
S100E in rats; Subheading
3.4
the setup of the light-damage
model of induced retinal degeneration in rats; Subheading
3.5
the evaluation of retinal function by full-field electroretinogram
(ERG) recordings; Subheading
3.6
biological sample (blood,
serum, anterior chamber fluid (ACF), and eye) collection and
hematocrit measurement; Subheading
3.7
the quantification of
EPO and S100E protein levels in biological fluids of rats;
Subheading
3.8
the evaluation of rat photoreceptor survival by
retinal histology.
The detailed methods and protocols for AAV vector production
are described in “
Viral Vectors for Gene Therapy
.
Methods and
Protocols
” (
49
). Briefly, AAV vectors can be produced at high yields
by transient triple transfection of mammalian cells or infection of
packaging eukaryotic and insect cells (
49
). Independently of the
specific method used for AAV vector production, it is necessary to
generate a
cis
plasmid that contains the expression cassette with the
gene of interest flanked by the viral inverted terminal repeats (ITRs,
145 bp). The ITRs are the only viral sequences required in
cis
to
allow the encapsidation of the AAV vector genomes for the pro-
duction of viable vector particles during the AAV vector produc-
tion process (Fig.
2
).
3.1 The Generation
of Plasmids for AAV
Vector Production
Including Expression
Cassettes Encoding
for EPO and the EPO
Derivative S100E
3.1.1 Basics of AAV
Vector Production
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