Plenti3.3 tr

We generated a ViraPower^TM T-Rex^TM OKF6/TERT-1 cell line through the transduction of pLenti3.3/TR (Life Technologies). The all-in-one cassettes were subcloned into a pLenti6.3/TO/V5-DEST vector (Life Technologies). After we transduced a ViraPower^TM T-Rex^TM OKF6/TERT-1 cell line with pLenti6.3/TO/V5-DEST vectors, we selected four PAX6-a/OCT4/KLF4- and PAX6-b/OCT4/KLF4-inducible colonies.

Expression constructs that contain sequences that code for EAV nsp2 and nsp3 were assembled in pDONR201 (Life Technologies, Inc.) with an HA tag at the N terminus of nsp2 and enhanced GFP (eGFP) fused to the C terminus of nsp3. The pDONR construct was then transferred using LR Clonase II (Life Technologies, Inc.) to either pcDNA3.1-DEST or pLenti6.3/TO/V5-DEST (Life Technologies, Inc.). Helper plasmids for lentivirus particle production have been described previously (51 (link)), and pLenti3.3/TR carrying the tetR gene was purchased from Life Technologies, Inc. (34 (link)). To make CRISPR/Cas9 knockout cell lines, the LentiCrisprv2 vector was used as previously described (52 (link), 53 (link)). Guide RNA sequences are listed in Table S1 in the supplemental material (54 (link)).

Primary astrocytes were prepared from one-day-old neonatal rat or mouse [39 (link)]. Mouse embryonic fibroblasts (MEFs) were prepared from 13.5–14.5 day old fetuses [40 (link)]. Cells were maintained at 37°C under 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% bovine calf serum (BCS), 50 units/ml penicillin, 90 units/ml streptomycin. All cell cultures were passaged every 5–7 days for up to 3 passages.
PC3, HEK293, HeLa and C8D1A cells were maintained in DMEM supplemented with 25 mM HEPES (pH 7.4) 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine. Cells were seeded at a density of 15,000 cells/cm² 24 h prior to transfection.
Transient transfections were done with Fugene6 according to manufacturer’s instructions. Cell lysates were prepared 48 h after transfection. Each transfection experiment repeated at least 3 times and each assay point was determined in triplicate.
Tunable ectopic protein(s) expression was done using the Tet-ON expression system (Life Technologies). The Tet Repressor Protein (TR) was integrated in the genome of PC3 cells by lentivirus infection (pLenti3.3/TR, Life Technologies) and cell selected with G418. TR expressing PC3 cells were transfected with pTREX-DKK3-HA or pTREX-Flag-IBS and ectopic protein expression induced by addition of increasing concentrations of doxycycline to the growth medium.