Biology Reference
In-Depth Information
The EGFP CDS and the regulatory elements can be easily
exchanged with the gene of interest and other regulatory
elements, respectively.
2. To perform the S100E site-directed mutagenesis we used
primers that contained a nucleotide triplet (e.g., GAA) corre-
sponding to glutamate, thus resulting in the substitution of the
triplet encoding serine in the S100E PCR product. In the wild
type EPO CDS the AGT triplet encoding serine is located at
the amino acidic position 126 that corresponds to the amino
acidic position 100 in the mature EPO protein (devoid of the
hormone signal peptide).
3. The transfection of the plasmids encoding EPO and S100E in
cell lines other than COS7 is also feasible, however it is possible
that the EPO and S100E glycosylation profile (and their appar-
ent molecular weight) may vary slightly between different cell
lines.
4. AAV vectors are handled as biosafety level 2 genetically-
modified micro-organisms in several countries. Please observe
the legislation regarding biosafety levels operating procedures
of the country where you plan to perform the experiments
using AAV vectors.
5. For the optimal storage of AAV vectors store them in aliquots
at −80°C in 5% sterile glycerol.
6. No reduction of viral infectivity is observed up to eight sequen-
tial freeze and thaw events (Doria M. and Auricchio A,
unpublished data). However we suggest using a freshly thawed
aliquot each time.
7. It is possible to use albino rat strains other than Lewis (e.g.,
Sprague Dawley or Wistar) but it is important to consider that
the genetics as well as the age and the diet can affect the degree
of damage induced by light.
8. During light damage, animals need to have free access to food
and water.
9. To compare the protective effect of a specific treatment from
light damage it is important to use an adequate number of
sham-treated controls. In particular, it is important to have a
sham-treated control per light damage cycle to evaluate the
variability of the damage that mainly depends on the lumi-
nance variability and the rat behavior (e.g., sleeping time and
eyes position with respect to the light sources).
10. The retinal light damage varies also with light history, circadian
rhythm, and the prior extent of dark-adaptation (
50
). Therefore,
to reduce the light-damage variability between groups of rats
undergoing distinct light-damage cycles it is important to start
each light damage cycle at a fixed time of day.
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