Photoreceptor Degeneration in Mice: Adeno-Associated Viral Vector-Mediated Delivery of Erythropoietin - Tissue-Protective Cytokines: Methods and Protocols - page 230

non-erythropoietic EPO-Ds protects PRs from induced ( 46 ) and
inherited ( 46, 47 ) degeneration.
Based on our study ( 46 ) we describe a method to deliver EPO
and the EPO derivative S100E (S100E) in the rat model of light
damage by AAV vectors and to evaluate the protective effects of
EPO and S100E upon PR function and survival.
In addition, this method may be useful to study the mecha-
nisms responsible for the protective effects induced by EPO and
EPO-Ds in the retina.

2 Materials

1. A plasmid for AAV vector production containing the AAV2
inverted terminal repeats (ITRs) (e.g., the pAAV2.1-CMV-
EGFP ( 48 ) that can be supplied by the author) (see Note 1).
2. Murine cDNA (e.g., from kidney, retina, or other tissues
expressing EPO).
3. Sequence-specific forward and reverse oligonucleotides for the
amplification of the EPO coding sequence (CDS). The oligo-
nucleotides must contain the restriction sites for the directional
cloning of the EPO CDS into the pAAV2.1-CMV-EGFP plas-
mid (e.g., NotI and BamHI).
4. Standard equipment and reagents for PCR amplification.
5. Standard equipment for DNA electrophoresis.
6. Standard equipment and reagents for DNA gel extraction and
PCR purification.
7. Standard equipment and reagents for: molecular cloning (e.g.,
restriction enzymes and DNA ligase); bacterial transformation
(e.g., Escherichia coli DH5

2.1 Generation
of Plasmids for AAV
Vector Production
Expressing EPO
and S100E

cells), growth (e.g., LB medium),
and selection (e.g., Ampicillin); low-scale plasmid purification
reagents.
8. Quick Change II XL Site-Directed Mutagenesis kit (Agilent
Technologies, USA).
9. Sequence-specific forward and reverse oligonucleotides to gen-
erate the S100E mutant by EPO mutagenesis (see Note 2).
10. SmaI and SnabI restriction enzymes.
11. Endotoxin-free plasmid purification materials (e.g., Endofree
Plasmid Giga kit, Qiagen, USA).

α

2.2 EPO and S100E
Protein Expression
In Vitro

1. COS7 cells (see Note 3).
2. Standard equipment and reagents for cell cultures.
3. Culture medium: Dulbecco's Modi fied Eagle's Medium (DMEM,
high glucose) containing 10% of Fetal Bovine Serum (FBS),
2 mM l-Glutamine and 1× antibiotic-antimycotic solution.
4. PolyFect Transfection Reagent (Qiagen).

2.2.1 Cell Culture
and Transfection