Lentiviral Vectors for Gene Delivery to the Nervous System - Gene Delivery and Therapy for Neurological Disorders - page 52

the titer as TU per ml of vector sample; at step 3 you have
prepared your vector dilutions at fi nal volume 500 ʼ l and from

l to your cells) [ 121 ].
19. The titer should be calculated using a percentage of fl uores-
cent positive cells within the range of 1-20 %. At this range,
the chance for a single cell to be infected by more than one
virus is small, thus reduce the possibility of underestimating
the titer ( see Note 11 ).

that you have applied 450 ʼ

1. Prepare serial dilutions of the vector samples. For unconcen-
trated samples use dilutions from 10 −4 to 10 −6 , while for con-
centrated ones from 10 −6 to 10 −8 [ 121 ].
2. Proceed to the determination of the p24 levels in the diluted
vector samples using a Retro-Tek HIV-1 p24 antigen ELISA
kit (we recommend Zeptometrix). Follow the manufacturer's
instructions to complete the assay ( see Notes 12 and 13 ).

3.3.2 Titration by
p24 ELISA

To accurately determine transducing titers of LVs produced, when
fl ow cytometry or other detection methods are not possible, qRT-
PCR assays can be used.

3.3.3 Titration by
qRT-PCR

There are several kits available, which employ quick RNA purifi ca-
tion step and determine viral RNA genome content using qRT-PCR
and SYBR technologies. We recommend the Lenti-X qRT-PCR
titration kit (Clontech), which is designed for all HIV-1 based vec-
tors and provides a fast and simple method for titrating LV stocks
( see Note 14 ). The kit allows target cell infection with vector prep-
arations harvested on the same day, thus the freeze-thaw cycles
that reduce the vector infectivity can be avoided, though we rec-
ommend titrating vectors that have already been frozen in order to
have a realistic titer of your vector preparation since all vector
stocks are preserved at −80 °C.
In this assay, viral supernatant is collected from virus produc-
ing cells (unconcentrated and concentrated viral preparations).
Concentrated viral vector is diluted 200-300-fold with cell culture
medium (i.e., mix 1

Assay to Determine
Relative Vector Particle
Numbers Based on Virion
RNA (One-Step qRT-PCR)

l of
medium) and using a viral RNA purifi cation kit, genomic viral
RNA is purifi ed. A DNase treatment removes any residual plasmid
DNA that may have been carried over from the transient transfec-
tion of HEK 293T cells during production. Serial dilutions of the
viral RNA are prepared and subjected to qRT-PCR to determine
the threshold ( C t ) values for each dilution. Samples are amplifi ed
alongside a RNA standard to correct for low level transcriptase
activity ( see Note 15 ). A standard curve is generated from serial
dilutions of a RNA control template. The copy number that cor-
responds to its C t value on the standard curve is used to calculate
the RNA genome copy number in a sample ( see Note 16 ).

ʼ l of concentrated vector stock and 199 ʼ