Mekkaoui, L;
Bentley, EM;
Ferrari, M;
Lamb, K;
Ward, K;
Karattil, R;
Akbar, Z;
... Pule, M; + view all
(2021)
Incorrect DOI - please do not claim.
Bio-protocol
, 11
(16)
, Article e4128. 10.21769/BioProtoc.4128.
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Abstract
The use of recombinant lentivirus pseudotyped with the coronavirus Spike protein of SARS-CoV-2 would circumvent the requirement of biosafety-level 3 (BSL-3) containment facilities for the handling of SARS-CoV-2 viruses. Herein, we describe a fast and reliable protocol for the transient production of lentiviruses pseudotyped with SARS-CoV-2 Spike (CoV-2 S) proteins and green fluorescent protein (GFP) reporters. The virus titer is determined by the GFP reporter (fluorescent) expression with a flow cytometer. High titers (>1.00 E+06 infectious units/ml) are produced using codon-optimized CoV-2 S, harbouring the prevalent D614G mutation and lacking its ER retention signal. Enhanced and consistent cell entry is achieved by using permissive HEK293T/17 cells that were genetically engineered to stably express the SARS-CoV-2 human receptor ACE2 along with the cell surface protease TMPRSS2 required for efficient fusion. For the widespread use of this protocol, its reagents have been made publicly available.
| Type: | Article |
|---|---|
| Title: | Incorrect DOI - please do not claim |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.21769/BioProtoc.4128 |
| Publisher version: | https://doi.org/10.21769/BioProtoc.4128 |
| Language: | English |
| Additional information: | This version is the version of record. For information on re-use, please refer to the publisher’s terms and conditions. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute > Research Department of Haematology |
| URI: | https://discovery-pp.ucl.ac.uk/id/eprint/10191304 |
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