Lenti x hek293t

Baby Hamster Kidney cells (BHK-21, Kerafast, Cat # EH1011), Lenti-X Human Embryonic Kidney cells (Lenti-X HEK-293T, Clontech, Cat # 632180), Normal Human Bronchial Epithelial cells (NHBE, Lonza, Cat # CC-2541) and HEp-2 cells (ATCC, Cat # CCL-23) were maintained in the respective growth medium and incubated at 37°C, 5% CO₂ atmosphere and 95% relative humidity.
BHK-T7 cells constitutively expressing T7-RNA polymerase were developed using lentiviral transduction. Cells were used for the production of the rgRSV-P-BlaM recombinant virus. BHK-21, BHK-T7 and Lenti-X HEK-293T were grown in DMEM supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) (Gibco BRL, Carlsbad, CA, United States), 100 U/mL penicillin, 100 μg/mL streptomycin. BHK-21 and BHK-T7 required an additional supplement of 2 mM L-glutamine; while the culture medium of Lenti-X HEK-293T required to be supplemented with 1 mM sodium pyruvate.
HEp-2 Cells were obtained from American Type Culture Collection (ATCC number: CCL-23) and maintained in Opti-MEM (Gibco BRL, Carlsbad, CA, United States) supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin.
Undifferentiated bronchial epithelial cells (NHBE) were maintained in Bronchial Epithelial Growth Medium (BEGM) (Lonza, Walkersville, MD, United States) following the supplier’s instructions.

To produce lentivirus, 450 000 Lenti-X HEK293T (Clontech) cells were seeded per well of a six-well plate and co-transfected with 2.8 µg pHAGE-mKeima-LGALS3 (Addgene; 175780), 2.3 µg pPAX2 (Addgene; 12260) and 0.85 µg pMD2G (Addgene; 122259) using Lipofectamine 2000 (Invitrogen; 1668027), according to the manufacturer's protocol. After 48 h, the virus-containing media were harvested. To determine the viral titre and generate stably expressing cells, RPE1 Flp-In VPS35 WT and (D620N) cells were transduced with mKeima-LGALS3 (Gal3) using polybrene (8 µg/ml) and after 24 h, subjected to puromycin selection (1 µg/ml). The mixed pools of puromycin-resistant cells were used for experiments.