Biology Reference
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11. The amount of cellular lysate to be analyzed by SDS-PAGE
may vary from 10 to 50
g per sample depending on the trans-
fection efficiency. We suggest loading at least two different
dilutions of each sample to ensure the clear detection of the
EPO and S100E proteins.
12. The murine EPO protein has a molecular mass of 30.4 kDa,
however, due to its secondary modifications (glycosylation), it
migrates with a higher apparent size of 34-38 kDa on SDS-
PAGE.
13. Several academic and nonacademic providers sell AAV vector
preparations. The only material that the applicant has to pro-
vide is a certain amount of the
cis
AAV plasmid containing the
gene of interest (Fig.
2
). The AAV vector core facility operat-
ing at the Telethon Institute of Genetics and Medicine
(TIGEM) requires 1.3 mg of endotoxin-free pAAV2.1 plasmid
for the production of an AAV preparation (4 mL of vector
preparation containing
μ
10e12 AAV genome copies).
14. Before providing the plasmid containing the transgene of inter-
est to the facility it is important to check the integrity of the
AAV ITRs that flank the transgene expression cassette. The
AAV ITRs are palindromes difficult to sequence unambigu-
ously. Thus, the assessment of ITR integrity is usually based on
the analysis of their restriction pattern. In the pAAV2.1 plas-
mid a fragment of 125 bp containing the AAV2 ITRs can be
excised by digestion of pAAV2.1 with the SmaI and SnabI
restriction enzymes.
15. In our experiments in the light damage rat model of photore-
ceptor degeneration we used AAV serotype 1 (AAV2/1)
because it is very efficient at transducing both muscle and the
retinal pigment epithelium (RPE), which we used for systemic
and intraocular expression of EPO and its derivatives, respec-
tively (
48, 51
).
The intramuscular injection of AAV-EPO in rats at the dose of
1 × 10
11
GC determines significant mortality at about 3 weeks
following the injections. This is probably due to the extremely
high increase in hematocrits that is caused by the systemic
overexpression of EPO. The mortality rate of rats injected
intramuscularly with AAV-EPO can be significantly reduced
using a tenfold lower AAV-EPO dose (1 × 10
10
GC/per rat).
17. The surgical procedures have to be performed in accordance
with the Association for Research in Vision and Ophthalmology
Statement for the Use of Animals in Ophthalmic and Vision
Research and with the specific country regulations.
18. For unambiguous identification number the rats before the
injections using ear punches or toe clipping.
≥
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