Lenti x gostix

The lentiviral expression construct pScalps_TRPML1-mCherry-HA and pScalps_CEPIA2mt was generated by molecular cloning using pCMV-TRPML1-mCherry-HA (gift from Shmuel Muallem) or pCMV CEPIA2mt (gift from Masamitsu Iino, Addgene plasmid no. 58218) as a PCR template. The insert was ligated into BamHI/Not restriction site of pScalps_Puro (gift from Silvia Monticelli, Addgene plasmid no. 99636). Pseudotyped lentiviral particles were produced by cotransfecting LentiX 293T cells (Takara Bio) with pScalps_TRPML1-mCherry-HA or pScalps_CEPIA2mt, packaging vectors pRSV-Rev and pMDLg/pRRE (gifts from Didier Trono, Addgene plasmid nos. 12253 and 12251), and pCMV-VSV-G (gift from Bob Weinberg, Addgene plasmid no. 8454) using Lipofectamine 3000 (Invitrogen). Virus-containing media were harvested and filtrated 48 h after transfection. The titer was determined using LentiX GoStix (Takara Bio). Virus was concentrated using LentiX concentrator (Takara Bio) and stored at −80°C until use.

Plasmids for lentivirus-mediated gene expression, pCAG-HIVgp, pCMV-VSV-G-RSV-Rev and pCSII-EF-MCS, were obtained from Dr. H. Miyoshi at RIKEN Tsukuba Institute. The PCR-amplified cDNA fragment for wild-type human KIF11 was cloned into pCSII-EF-MCS, and G268dV [8 (link)] or S1017fs mutation (this study) was introduced [11 (link)]. Lentivirus packaging was conducted as described (http://cfm.brc.riken.jp/lentiviral-vectors/protocols/). Briefly, 17 μg of pCSII-EF-MCS-based plasmid containing KIF11^WT (± 3×HA tag), KIF11^G268V or KIF11^S1017fs (±3×HA tag) was co-transfected with 10 μg each of the packaging plasmid pCAG-HIVgp and the VSV-G/Rev-expressing plasmid pCMV-VSV-G-RSV-Rev into 293T cells using the calcium phosphate co-precipitation method. The medium was replaced after 16 h of transfection and the cells were cultured for a further 48 h. Viral particles were concentrated using Lenti-X-Concentrator (#631231, Takara Bio, Kusatsu, Japan) and the titer was measured with Lenti-X-GoStix (#631243, Takara Bio). U-2OS cells or HeLa cells were transduced at a MOI of 1 or 10, and stable clones were obtained by limiting dilution.

Lenti-X 293 T cells (2 × 10⁶ cells/dish) were plated in a BioCoat Collagen I Cellware 35 mm dish (BD Biosciences, Franklin Lakes, NJ, USA) the day before transfection. DNA transfection was performed using the Lenti-X HTX Packaging System (Takara) with phyp53-HA-LVSIN or pGFP-LVSIN according to the manufacturer’s instruction. After 2 days of incubation at 37 °C, the lentivirus vector-containing supernatant was harvested. Lenti-X GoStix (Takara) were used to rapidly confirm the presence of lentivirus. The lentivirus containing supernatant was transferred to the BioCoat Fibronectin 35 mm dish (BD Biosciences) in order for the lentivirus vector to bind to the fibronectin coating on the dish. After a 5-h incubation at 37 °C, HEK293 cells (with 8 μg/mL hexadimethrine bromide, Sigma-Aldrich) were plated to the lentivirus vector-bound dish and incubated for 2 days at 37 °C. The vector-transduced cells were selected by culturing the cells in G418 (800 μg/mL, Nacalai Tesque)-containing medium. After 7 days, the cells were harvested and the expression of hyp53-HA protein was confirmed by western blotting as mentioned above.

HEK293T cells were cultured in RPMI complete medium at 37°C in 5% CO₂. When cell growth reached 70-80% confluence, cells were transfected with the reconstructed LentiCRISPRv2-mCherry vector and two additional vectors: lentiviral packaging plasmid (pCMV-dR8.91, Addgene) that expresses HIV structural and packaging genes and envelope plasmid (pCMV-VSVG, Addgene) that expresses the pseudotyping envelope protein Vesicular Stomatitis Virus Glycoprotein (VSVG). Sixteen hours later, the media was removed from the transfected cells and replaced with 5% RPMI complete medium (RPMI Medium supplemented with 5% heat-inactivated FBS and 100 IU/ml penicillin and 100 μg/ml streptomycin). After 24 h, the supernatant containing lentiviral particles was collected and filtered through 0.45 µm pore size membranes and stored at 4°C (the first collection). An additional fresh 5% RPMI complete medium was added to the cell culture flask and cultured for another 24 h. The supernatant was collected as described above (the second collection) and then combined with the supernatant in the first collection and concentrated using a Lenti-X concentrator (Takara, Melbourne, Australia). Virion titer was measured by Lenti-X-GoStix (Takara) to establish the presence of functional virions at > 5 x 10⁵ infective units per ml (IFU/ml). The generated lentiviral particles were aliquoted and stored at -80°C before use.

Lentiviral production was confirmed with Lenti-X GoStix (Takara) and lentiviruses were concentrated with Lenti-X Concentrator (Takara) according to the manufacturer’s recommendations. For transduction, 1 × 10⁶ NALM-6 cells in 500 µl of medium were added to the concentrated virus. 5 µg/ml polybrene (Sigma-Aldrich) was added. The cells were centrifuged at 800 g and 32 °C for 30 min. Cells were then transferred into 6-well plates and cultivated in normal growth medium without antibiotics. Selection was started after 48 h with 0.5 µg/ml puromycin (Thermo Fisher Scientific). Antibiotic medium was exchanged every 2–3 days. As soon as cells were not dying under selection anymore and the population was stable, induction experiments were started. After transduction, cells remained in the S2 laboratory for at least 6 weeks. Then, Lenti-X GoStix was used to check for any remaining lentivirus.