Biomedical Engineering Reference
In-Depth Information
Sixty hours after transfection, visually compare the proportion/
level of transfection by fluorescent microscopy to determine
the optimal volume for transfection.
6. Other factors which may influence AAV vector yield include
insert size, for example, inserts that exceed the optimal packag-
ing size of 4.1-4.9 Kb are packaged with lower efficiency and
the generated vectors may not be functional. Toxicity as a
result of the overexpression of a cytotoxic transgene can also
attenuate vector titer. The effect of transgene toxicity on
HEK293 cells can be reduced by plating cells at a slightly
higher density (~80 %) and by reducing the amount of pAAV
expression plasmid by 50-75 %.
7. We use primers designed to amplify a specific 120 bp region of
the WPRE common to all our rAAV expression cassettes.
Primers can also be designed to other elements, such as pro-
moters and polyadenylation signals, as long as they are present
in both the plasmids used to construct the standard curve, the
titering control vector, and unknown vector samples.
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