Lentivirus RT-PCR

1. Seed 293T cells were seeded in a two-tray Nunc A/S cell factory (CF2) at 60x10^6 cells per cell factory in OPTIMEM with glutamax supplemented with 2% FCS and 20 μg/ml gentamicin.

2. Next day, an 11.2 ml mixture of the three plasmids was made in 150 mM NaCl: 320 μg of transfer plasmid,
160 μg of packaging construct and 80 μg of envelope plasmid. An equal volume of PEI solution was added slowly to this DNA mixture and the solution was incubated at room temperature for 15 minutes. The DNA-PEI complexes were added to 150 ml OPTIMEM without FCS and added slowly to the 293T cells for 24 hrs. Supernatant was harvested 2 and 3 days post-transfection and filtered through a 0.45 μm easy-flow filter (Becton Dickinson Labware, Le pont de Claix, France) and further processed as described before. Apart from production in a CF2, we also produced vectors in a 10-layer cell factory (CF10), containing 500 ml medium. DNA concentrations for transfection are 3200 μg transfer plasmid, 1600 μg packaging plasmid and 800 μg envelope plasmid for 2 CF10. The DNA concentrations were adapted proportionally to the surface area.

RNA content of lentiviral vectors was obtained by a one-step RT-PCR which allows reverse transcription
(RT) and amplification in the same reaction tube. After RNA extractions on concentrated vector preps
with the RNAqueous®-Micro Kit (Ambion, Cambridgeshire, United Kingdom) a DNase treatment (RNase
free DNase 1U/ l, Promega, Leiden, The Netherlands) was carried out to eliminate residual plasmid
DNA from the vector production. RNA was amplified using the TaqMan® One-Step RT-PCR master mix
reagents kit (Applied Biosystems, Lennik, Belgium) with primers and probe located in the eGFP
transgene, the LTR or the WPRE.
Forward and reverse primers were developed with the Primer
Express Software (Applied Biosystems) and are specific for eGFP: 5’- GGAGCGCACGATCTTCTTCA-
3’
and
5’-AGGGTGTCGCCCTCGAA-3’;
for
LTR
5'-TGTGTGCCCGTCTGTTGTGT-3'
and
5'-
GAGTCCTGCGTCGAGAGAGC-3' [130]; and for WPRE 5'-CCGTTGTCAGGCAACGTG-3' and 5'-
AGCTGACAGGTGGTGGCAAT-3' [81].
TaqMan probes used are for eGFP: 5’-FAM-CTACAAGACCCGCGCCGAGGTG-TAMRA-3’; for LTR: 5'-
FAM-CAGTGGCGCCCGAACAGGGA-TAMRA-3'
[130]
TGCTGACGCAACCCCCACTGGT-TAMRA-3' [81].
and
for
WPRE:
5'-FAM-
The kit contains the MultiscribeTM Reverse
Transcriptase (MuLV) which carries out the RT-step (30 minutes at 48°C) and AmpliTaq Gold enzyme
for amplification (40 cycli at 95°C for 15 seconds, followed by 1 minute at 60°C). Reactions were
analyzed using the ABI Prism model 7700 sequence detection system (Applied Biosystems). During
each run a ‘no amplification control’ (NAC) was included for every sample (sample without RT-enzyme)
to detect residual DNA contaminants and this value was subtracted. To normalize the mRNA values of
expressed transgenes to the total RNA content in the sample, additional primers and a VIC-labeled
probe directed against the RNAse P housekeeping gene were included in the sample mixture (RNAse P
control reagents kit, Applied Biosystems) and values were corrected to the total amount of RNA in the
sample.