Lentiviral construct coding for LMNA R321X mCherry-tagged protein was produced by Stratagene's Quik Change II XL site-directed mutagenesis kit (Agilent Technologies), using the the lentiviral plasmid encoding for mCherry-tagged Lamin WT mCherry-tagged (pLV[Exp]-Neo-CMV > mCherry(ns):hLMNA[NM_170707.4], Vector Builder). Mutagenic primers were designed using Quik Change Primer Design Program as previously shown [17 (link)]. The mutation was verified by sequencing.
Viral particles were produced in HEK-293T cells by simultaneously co-transfection (Invitrogen™ Lipofectamine™ 2000 Transfection Reagent) of the plasmid carrying the gene for protein of interest (mCherry-tagged-Lamin WT or mCherry-tagged-LMNA R321X), the envelope plasmid encoding VSV-G and the packaging plasmid encoding Gag/Pol and Rev, as previously reported [13 (link)].
For lentiviral transduction, HL-1 cells were plated in a 6-well plate at 30–50% of confluency, and after adhesion, they were incubated with 500 µL of virus-containing medium at 37 °C in a humidified 5% CO2 incubator for 18 h. After viral particles removal, cells were then incubated with 400 μg/mL Geneticin (G418, Gibco, Life Technologies) for 1 week to select stable clones. Pure clones were selected by fluorescence microscopy.
Viral particles were produced in HEK-293T cells by simultaneously co-transfection (Invitrogen™ Lipofectamine™ 2000 Transfection Reagent) of the plasmid carrying the gene for protein of interest (mCherry-tagged-Lamin WT or mCherry-tagged-LMNA R321X), the envelope plasmid encoding VSV-G and the packaging plasmid encoding Gag/Pol and Rev, as previously reported [13 (link)].
For lentiviral transduction, HL-1 cells were plated in a 6-well plate at 30–50% of confluency, and after adhesion, they were incubated with 500 µL of virus-containing medium at 37 °C in a humidified 5% CO2 incubator for 18 h. After viral particles removal, cells were then incubated with 400 μg/mL Geneticin (G418, Gibco, Life Technologies) for 1 week to select stable clones. Pure clones were selected by fluorescence microscopy.
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