Generation of Stable Expression Mammalian Cell Lines Using Lentivirus

Abstract

Lentiviruses are used very widely to generate stable expression mammalian cell lines. They are used for both gene down-regulation (by using shRNA) or for gene up-regulation (by using ORF of gene of interest). The technique of generating stable cell lines using 3rd generation lentivirus is very robust and it typically takes about 1-2 weeks to get stable expression for most mammalian cell lines. The advantage of using the 3rd generation lentivirus are that are very safe and they are replication incompetent.

Psychedelic-inspired drug discovery using an engineered biosensor

“…After 14 h of ll Cell 184, 2779-2792.e1-e11, May 13, 2021 e7 incubation, the medium was exchanged for fresh DMEM. After an additional 48 h of incubation, the lentivirus-containing medium was collected, filtered through a 0.45 mm Durapore low-protein binding filter, concentrated using a Centricon-70 ultra filtration unit at 3,500 g for 50 min, and stored at À80 C. Next, confluent HEK293T cells that had been grown in 24-well plates were infected with 20 mL of concentrated lentivirus for 48 h. Puromycin selection was performed as described by Tandon and co-workers (Tandon et al, 2018). Expression was assessed via fluorescence microscopy, and a single cell was selected for expansion.…”

Evaluating an In-House Cell-Based Assay for Detecting Antibodies Against Muscle-Specific Tyrosine Kinase in Myasthenia Gravis

“… 11 21 22 Transient transfection can be performed both easily and rapidly, but it has limitations in the duration of protein expression being short and the transfection needing to be conducted repeatedly. In contrast, a gene of interest could be integrated into the host genome by using a lentivirus, and this method has previously been shown to be efficient and reliable for generating stable cell lines, 23 as demonstrated by the use of a lentivirus to generate a cell line that stably expresses MuSK protein in the present study.…”

Aberrant expression of embryonic mesendoderm factor MESP1 promotes tumorigenesis

“…p19 ARF null MEFs were grown in a 9% CO 2 incubator. MESP1-knockdown and Mesp1-overexpressing stable cell lines were generated using lentiviruses as described in previously published protocol [17]. In order to establish doxycycline-inducible Mesp1-V5 expression system, cells were incubated with 1 μg/ml of doxycycline (Sigma) for 15–20 days with fresh doxycycline being replenished every 48 h.…”