Biology Reference
In-Depth Information
5. Other ratios in the packaging plasmids may be used, following
the producer/provider instructions.
6. The transfection efficiency is the limiting step in determining
the final titer of the virus production. Among the different
parameters controlling the transfection efficiency, two critical
parameters to be carefully taken into account are the pH and
the reaction time as they regulate the formation and growth of
DNA-calcium phosphate precipitates [ 89 ]. Regarding pH, it
is critical to identify the optimal pH of the 2× HBS solution,
depending on the specific conditions (pH meter, temperature,
etc.). We thus suggest testing at least three different batches of
2× HBS with three slightly different pH (e.g., between 7.08
and 7.15) in order to identify the best conditions for transfec-
tion. In addition to this, the time period allowed for the gen-
eration of the precipitates is another important parameter.
Subsequent to binding of most of the soluble DNA into pre-
cipitates, particles tend to grow further and transfection effi-
ciency will be reduced. It is then fundamental to add the
solution containing the precipitates immediately to the culture
dish.
7. During the lentivirus collection and concentration, take all the
safety measures necessary to avoid spreading of infective viral
particles and potential infection. Refer to the Health and
Safety guidelines of your institution for details. General safety
measures to be adopted include wearing disposable lab coats,
double gloves, and overshoes. Moreover, sterilize all the mate-
rials (pipettes, tips, culture dishes, tubes, etc.) coming into
contact with the viral particles by cleaning them with an appro-
priate disinfectant agent (e.g., bleach or commercial disinfec-
tants) and subsequently by autoclaving. No sharp objects
should be used at any time.
8. The number of dishes to be used mainly depends on the vol-
ume and titer of virus you wish to obtain and on the capacity
of the ultracentrifuge rotor. For example, using a rotor with a
capacity of 210 ml you can transfect 14 dishes and finally
obtain 420 μl of virus (concentrated 500×) with a titer of 10 8 -
10 9 TU/ml.
9. It is important to seed NPCs at high density before infection.
This step is critical for exposing the maximum number of cells
to the vector while at the same time maintaining conditions
that allow rapid re-expansion of the infected cell population.
10. As the MOI increases, the percentages of cells infected with at
least one viral particle also increases. MOI of 10 might be used
because it assures that essentially every cell is infected. There
are substantial differences in cell types that affect susceptibility
to infection. High MOI is used when the experiment requires
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