All lentiviral vectors were handled in a class II biosafety laboratory. All plasmids were purchased on Addgene: pTet-O-Ngn2-puro (Addgene ID: 52047), M2-rtTA (Addgene ID: 20342), pRSV-REV (Addgene, #12253), pMDLg/pRRE (Addgene, #12251), and pMD2.G (Addgene, #12259). Lentiviral particles were generated with CaCl2 transfection. For transfection, 22 μg of pMD2.G, 15 μg of pRSV-REV, 30 μg of pMDLg/pRRE and 75 μg of the lentiviral vector of interest (rtTA or Ngn2) were mixed in 2610 μl of TE buffer, then 290 μl of 2.5 M CaCl2 were added dropwise to the mixture and later 2900 μl of 2x HeBS buffer were added dropwise and incubated 5 min. Mixture was then added dropwise to two T175 flasks of HEK 293T cells (half of the mixture to each flask). Medium was changed 16 h after transfection and viruses were harvested 48 h after transfection, pelleted at 20,000 × g for 2 h at 4°C, resuspended in 100 μl DMEM overnight, aliquoted and kept at −80°C. Synapsin I-GFP lentivirus was kindly provided by Prof. Kokaia.
M2rtta
Manufactured by Addgene
M2rtTA is a reverse tetracycline-controlled transactivator protein. It binds to tetracycline operator sequences and activates transcription of target genes in the presence of tetracycline or its derivatives.
Automatically generated - may contain errors
3 protocols using m2rtta
1
Lentiviral Vector Production Protocol
2
Lentiviral Inducible Reprogramming Constructs
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The LVTHM-T7-Oct4, -Sox2, -cMyc, and -Klf4 constructs were described elsewhere (Liskovykh et al., 2011). LV-tTR-KRAB-iresBsd was derived from LV-tTR-KRAB-iresDsRed [66 (link)] by replacing the DsRed sequence with blasticidin S resistance gene. Other constructs were kindly provided by different labs: LV-tTR-KRAB-dsRed, pMD2.G, and psPAX2 [66 (link)], HAGE2-TetO-miniCMV-hOct4-F2A-hKlf4-IRES-hSox2-E2A-hcMyc-W-loxP (OKSM) [67 (link)], tetO-FUW-OSKM (OSKM, Addgene plasmid #20321) and FUW-M2rtTA (M2rtTA, Addgene plasmid # 20342) [33 (link)]. Lentiviruses were packaged in 293T cells using polyethylenimine hydrochloride (PEI 40 kDa, 40 μg) as a transfection method [68 (link)]. Lentivirus particles in cell culture supernatant were collected, concentrated to 5−10 × 106 TU/ml as described elsewhere [16 (link), 66 (link), 69 (link), 70 (link)].
3
Lentiviral-Mediated DYRK1A Overexpression
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A lentiviral vector system was used to generate SH-SY5Y sublines for controllable overexpression of GFP-DYRK1A or GFP-DYRK1A-K188R. In brief, rat DYRK1A and DYRK1A-K188R cDNA were amplified out of pEGFP-DYRK1A and integrated into the lentiviral FUW-tetON backbone (after removing the hMYC insert from Addgene plasmid 20723 with EcoRI) by homologous recombination in Escherichia coli,74 (link) resulting in FUW-tetON-GFP-DYRK1A and FUW-tetON-GFP-DYRK1A-K188R. Co-transduction of the FUW-tetON vector with FUW-M2rtTA (Addgene 20342), which encodes the tetracycline-dependent transactivator, allows for tetracycline-regulated expression of the transgene.75 (link) Lentivirus production was performed as described.30 (link) Co-transduction of SH-SY5Y cells then was titrated with different amounts of FUW-tetON-GFP-DYRK1A/KR and FUW-M2rtTA containing lentivirus until achieving maximal transduction efficiency as controlled by doxycycline-induced GFP fluorescence. These cell populations were considered as stably transduced, amplified, and frozen. All experiments were performed with cells derived from the same transduction experiment.
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