Biology Reference
In-Depth Information
effi ciency of LVs in the cells used for the test. However, permissive-
ness depends on LV pseudotyping and the transcriptional activity
of tissue-specifi c promoters strongly affects outcome.
This ELISA determines the concentration (ng p24/
l) of p24 cap-
sid in the samples. It is performed on nonconcentrated and con-
centrated LVs. During LV production, 5
μ
3.7.1 Determination of
the Physical Titer by ELISA
μ
l aliquots of supernatant
are taken and diluted in 45
l of the lysis buffer provided in the kit.
For concentrated LV-VSV-G, serial dilutions, from 1/10
6
to
1/1.2 × 10
7
, are prepared. For LVs pseudotyped with the MOK-G
envelope or containing an miRT sequence (lower yield,
see
Note
15
), the samples are diluted to 1/6 × 10
6
. Once diluted in lysis buf-
fer, LV samples can be transferred to a BSL1 (biosafety level 1)
laboratory. p24 antigen determinations are carried out in accor-
dance with the manufacturer's protocol (
see
Note 16
).
μ
This technique is used to determine the quantify lentiviral mRNA
copies following stable transduction in cell culture [
95
,
108
]. All
steps up to RNA extraction are performed in BSL2 or BSL3 labo-
ratories. The concentrated (1,000 times) viral suspension (2
3.7.2 Determination
of the Physical Titer
by RT-qPCR
μ
l) is
added to a 1.5-ml tube containing 353
μ
l DNase- and RNase-free
water, 5
l of DNaseI and 50 pg of RNaseA. The content of the
tube are mixed by vortexing and the tubes are then briefl y centri-
fuged. The mixture is incubated for 10 min at room temperature
and 20
μ
l of RNasin is then added. The mixture is incubated for
10 min at 37 °C and 10
μ
l of 25 mM EDTA for inactivation of the
enzyme by heating at 70 °C for 10 min. The tubes are then centri-
fuged at 2,000×
g
for 10 s. RNA is extracted with a Pure link RNA
miniKit (Ambion). For lysis, 300
μ
l of the lysis buffer provided in
the kit is added. At the end of the procedure, the purifi ed RNA is
eluted in 30
μ
μ
l of the kit solution (in RNase-free tubes). The puri-
fi ed RNA (5
l) is added to a 96-well plate for reverse transcription
with the Express Superscript mix and Express SYBR green ER
supermix (Invitrogen). As a negative control, the RNA is added to
the well without reverse transcriptase [
95
]. Finally, PCR is carried
out as follows: for 5 min at 50 °C, 2 min at 95 °C (denaturation),
and 40 cycles of 15 s at 95 °C (denaturation), and 1 min at 60 °C
(amplifi cation).
The primers used, the sequences of which are given below,
recognize the LTR of HIV-1 and can be used for the titration of
all LVs.
μ
Sense: TGTGTGCCCGTCTGTTGTGT
Antisense: GAGTCCTGCGTCGAGAGAGC
The number of RNA copies is obtained from a standard curve
for known numbers of copies of DNA plasmid treated in the same
way as the samples.
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