To produce lentiviral particles, plasmids encoding CERS5, CERS6, or
mCherry alone were co-transfected with packaging plasmids into Lenti-Pac 293 TA
cells (GeneCopoeia, Rockville, MD) grown on 100-mm dishes with Fugene 6 (Roche,
Branford, CT) according to manufacturer’s instructions. Forty-eight hours
later, culture medium was harvested and passed through a 0.45-micron filter to
remove cell debris. Viral stocks were used undiluted right away or aliquoted and
stored at −80°C.
To create MOLM-14 cells expressing CERS5, CERS6, or mCherry, cells were
dispensed on 12-well plates (Genesee Scientific, Morrisville, NC) at a density
of 500,000 per well in 0.1 ml of growth media. Lentiviral stocks were mixed with
Polybrene to final concentration of 8 ug/ml and added to cells in the amount of
1 ml per well. Plates were incubated at 37°C and 5% CO2 for 15
minutes, sealed with Parafilm and centrifuged at 25°C for 30 min at 1220
× g. After centrifugation, 3 ml of fresh growth medium was added to each
well. The next day, media was removed and infection repeated used 1 ml of fresh
lentiviral supernatant as described above.
Two or three days after infection, cells demonstrated a transduction
efficiency of 90% based on the mCherry signal, and were further sorted to obtain
a pure population of mCherry-positive cells. Expression of CERS5 or CERS6 was
confirmed by Western blotting.
mCherry alone were co-transfected with packaging plasmids into Lenti-Pac 293 TA
cells (GeneCopoeia, Rockville, MD) grown on 100-mm dishes with Fugene 6 (Roche,
Branford, CT) according to manufacturer’s instructions. Forty-eight hours
later, culture medium was harvested and passed through a 0.45-micron filter to
remove cell debris. Viral stocks were used undiluted right away or aliquoted and
stored at −80°C.
To create MOLM-14 cells expressing CERS5, CERS6, or mCherry, cells were
dispensed on 12-well plates (Genesee Scientific, Morrisville, NC) at a density
of 500,000 per well in 0.1 ml of growth media. Lentiviral stocks were mixed with
Polybrene to final concentration of 8 ug/ml and added to cells in the amount of
1 ml per well. Plates were incubated at 37°C and 5% CO2 for 15
minutes, sealed with Parafilm and centrifuged at 25°C for 30 min at 1220
× g. After centrifugation, 3 ml of fresh growth medium was added to each
well. The next day, media was removed and infection repeated used 1 ml of fresh
lentiviral supernatant as described above.
Two or three days after infection, cells demonstrated a transduction
efficiency of 90% based on the mCherry signal, and were further sorted to obtain
a pure population of mCherry-positive cells. Expression of CERS5 or CERS6 was
confirmed by Western blotting.