Generation of the pCMV6-AC-GFP lentiviral backbone expressing TurboGFP (OriGene #PS100010) and FL-PPARα or Y314D-PPARα was described before.10 (link),11 (link) Briefly, mouse PPARα ORF in pCMV6-AC-GFP vector (cat # MG 227641) was purchased from Origene followed by mutation at Tyr314 with aspartate (Y314D) by site-directed mutagenesis. To generate pLenti6.3/V5-TOPO® constructs of FL-PPARα or Y314D-PPARα, each construct was amplified by PCR followed by TOPO cloning reaction using Invitrogen kit (K5315–20) with pLenti6.3/V5-TOPO vector. One-Shot Stbl3 competent cells were used for transformation and sequencing of the clones was performed at ACGT Inc. Next, lentivirus production was carried out in 293FT cells using ViraPower™ Packaging Mix and pLenti expression plasmid DNA containing either FL-PPARα or Y314D-PPARα. Viral particles were concentrated with lenti-concentrator solution and MOI was calculated. During experiments, 10–12 days in vitro (DIV) PPARα−/− hippocampal neurons were transduced with lentiviral particles at MOI 10 for 48 h at 37°C. Live GFP imaging was used to monitor viral integration.
Kit k5315 20
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Generation of lentiviral constructs expressing PPARα variants
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Generation of lentiviral constructs expressing PPARα variants
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Generation of the pCMV6-AC-GFP lentiviral backbone expressing TurboGFP (OriGene #PS100010) and FL-PPARα or Y314D-PPARα was described before.10 (link),11 (link) Briefly, mouse PPARα ORF in pCMV6-AC-GFP vector (cat # MG 227641) was purchased from Origene followed by mutation at Tyr314 with aspartate (Y314D) by site-directed mutagenesis. To generate pLenti6.3/V5-TOPO® constructs of FL-PPARα or Y314D-PPARα, each construct was amplified by PCR followed by TOPO cloning reaction using Invitrogen kit (K5315–20) with pLenti6.3/V5-TOPO vector. One-Shot Stbl3 competent cells were used for transformation and sequencing of the clones was performed at ACGT Inc. Next, lentivirus production was carried out in 293FT cells using ViraPower™ Packaging Mix and pLenti expression plasmid DNA containing either FL-PPARα or Y314D-PPARα. Viral particles were concentrated with lenti-concentrator solution and MOI was calculated. During experiments, 10–12 days in vitro (DIV) PPARα−/− hippocampal neurons were transduced with lentiviral particles at MOI 10 for 48 h at 37°C. Live GFP imaging was used to monitor viral integration.
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