Hek293ft cells

Lentiviruses were generated as described previously with only slight modifications of the original protocol (Naldini et al., 1996 (link)). HEK293FT cells (Gibco; R700–07; RRID:CVCL_6911) plated on a poly-L-lysine (Sigma-Aldrich) coated 15 cm plastic dish were transfected with the packaging pVSV-G, the envelope pCMVdeltaR8.2, and the backbone vectors with Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. Cells were incubated with the transfection mix for 6 hr in Opti-MEM medium (Gibco) containing 10% fetal bovine serum (FBS, Gibco), and the medium was then changed to Dulbecco's Modified Eagle's Medium (DMEM, Gibco) containing 2% FBS, 10 units/mL penicillin (Gibco), 10 μg/mL streptomycin (Gibco), and 10 mM sodium butyrate (Merck). Forty-eight hours after transfection, the culture medium containing the lentiviral particles was harvested and concentrated using Amicon particle centrifugal filters (100 kDa; Millipore) according to the manufacturer's instructions. High-titer lentiviral samples were aliquoted, snap-frozen in liquid nitrogen, and stored at −80°C until used. HEK293FT cells, which were only used for virus production, were obtained from a low passage culture (P3) from the original cell line purchased from Gibco and checked for mycoplasma every 6 months.

HEK 293FT cells were purchased from Thermo Fisher Scientific (USA). MS-1 cells were purchased from ATCC (USA). Human Dermal Microvascular Endothelial Cells (HDMECs) were purchased from PromoCell (Germany). HEK293FT cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 2 mM L-Glutamine and 10% fetal bovine serum (all from Thermo fisher Scientific, USA). MS-1 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 2 mM L-Glutamine and 5% fetal bovine serum. HDMECs were cultured in Endothelial Cell Growth Medium MV2 with Endothelial Cell Growth Supplement (PromoCell, Germany). All cells have been tested negative for mycoplasma contamination. The identities of HEK 293FT and MS-1 haven’t been re-authenticated, but they have been used within limited passages upon purchasing.

Recombinant retroviruses were produced by transient transfection using HEK-293FT cells (Invitrogen). HEK-293FT cells were transfected with retroviral vector pMSCV MAPT (3RD-MAPT3) and plasmids encoding GAG-POL and VSV-G using lipofectamine 2000 (Invitrogen), according to the recommendation of the manufacturer.
The Rin-5F cells were used for the retroviral-mediated overexpression of MAPT. Semiconfluent Rin-5F cells were retrovirally transduced by spin infection (800 ×g, 90 min, 32°C) in the presence of polybrene (7 μg/mL). Four days after transduction, cells were treated with puromycin (1 μg/mL) for chemoselection. The culture supernatant was exchanged every third day and outgrowing cells were passaged every fourth day.
Sham-transfectant Rin-5F cells were obtained by transducing Rin-5F cells using empty pMSCV vector and chemoselecting for resistant cells with puromycin (1 μg/mL). This cell line was used for comparative reason and is named later an Empty cell line.

All cell lines were grown at 37°C, 5% CO₂. Human female embryonic kidney HEK293FT cells (#R70007; Invitrogen; RRID: CVCL_6911) were cultured in high‐glucose Dulbecco's Modified Eagle Medium (DMEM) (#41965–039; Gibco) supplemented with 10% fetal bovine serum (FBS) (#F7524; Sigma, or #S1810; Biowest) and 1% Penicillin–Streptomycin (#15140–122; Gibco).
HEK293FT cells were purchased from Invitrogen. The identity of the HEK293FT cells was validated by the Multiplex human Cell Line Authentication test (Multiplexion GmbH), which uses a single nucleotide polymorphism (SNP) typing approach, and was performed as described at www.multiplexion.de. All parental and edited cell lines were regularly tested for Mycoplasma contamination using a PCR‐based approach and were confirmed to be Mycoplasma‐free.