Bmal1 and UBC promoters were cloned into the pENTR-5’-TOPO vector (Life Technologies) as before17 (link). Mouse Bmal1, Bmal2 and various chimeras or mutants were first subcloned to p3xFlag-CMV-10 or -14 vectors to obtain 3xFlag tags at the N- or C-terminus, respectively, and then cloned to the pENTR/D-TOPO vector (Life Technologies). The promoter and cDNA pENTR vectors were recombined with pLV7 destination vector56 using Clonase (Life Technologies) to generate the lentiviral expression constructs. All constructs were verified by sequencing of the entire open reading frame.
Recombinant lentiviral particles were produced by transient transfection in human embryonic kidney HEK293T cells (ATCC) using the calcium-phosphate method as previously described56 ,57 (link). Bmal1–/–Per2Luc mouse fibroblast cell line was generated in our previous studies17 (link). Cells were cultured in DMEM supplemented with 10% FBS and 1x penicillin-streptomycin-glutamine mixture. All cell culture reagents were from HyClone. For infection of Bmal1–/–Per2Luc fibroblasts, culture medium containing viral particles (~106 viral particles/ml) were harvested at 48 hr post-transfection and used for subsequent infection of cells. Transduced cells were selected with 10 μg/ml blasticidin (InvivoGen) as previously done17 (link).
Recombinant lentiviral particles were produced by transient transfection in human embryonic kidney HEK293T cells (ATCC) using the calcium-phosphate method as previously described56 ,57 (link). Bmal1–/–Per2Luc mouse fibroblast cell line was generated in our previous studies17 (link). Cells were cultured in DMEM supplemented with 10% FBS and 1x penicillin-streptomycin-glutamine mixture. All cell culture reagents were from HyClone. For infection of Bmal1–/–Per2Luc fibroblasts, culture medium containing viral particles (~106 viral particles/ml) were harvested at 48 hr post-transfection and used for subsequent infection of cells. Transduced cells were selected with 10 μg/ml blasticidin (InvivoGen) as previously done17 (link).
