Lentiviral Vectors for Gene Delivery to the Nervous System - Gene Delivery and Therapy for Neurological Disorders - page 59

16. Poor standard curve is possibly caused by improper amplifi ca-
tion of DNA/RNA standards or inaccurate pipetting. Thus
avoid freezing and thawing of the standards and be precise
when pipetting tiny volumes.
17. Variations in TU, p24, and RNA titers can be attributed to the
variability of transient co-transfection used for vector produc-
tion, which depends strongly on the quality of producer cells
and the number of cells plated.
18. Incubation time in transduction experiments varies between
different applications. We routinely perform transduction for
6 h, following replacement with fresh medium. There are cases
where transduction is performed overnight or for 2-3 h. Once
again it depends on the cell line used.
19. Serum-free medium is recommended for transduction of pri-
mary cells. Immortalized cell lines can be transduced in com-
plete growth medium. Moreover, polybrene is omitted in
primary cell transduction as it has been reported to be toxic.
20. For transduction of cells in suspension, surfaces are usually coated
with poly- D -lysine and upon spinoculation cells are attached to
the bottom of the plate which allows easy medium renewal.

21. Spinoculation is suitable not only for cells in suspension, but
also for cells that are diffi cult to transduce, as spin infection
increases vector cell contact. In this case it is recommended to
perform a spin and non-spin transduction experiment in paral-
lel in order to determine the extent at which spinoculation
affects transduction effi ciency.

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