Vivapure adenopack 20 kit

Ad-VDR was generated using the Adeno-X Adenoviral System 3 kit (Takara #632269, Conda Laboratories, Madrid, Spain), following the manufacturer’s instructions. The VDR cDNA was obtained by RT-PCR from HeLa cells total RNA using the primers:

5′-gtaactataacggtcCACCCCTGGGCTCCACTTACC-3′ (forward)

5′-attacctctttctccCCGCCACAGGCTGTCCTAGTC-3′ (reverse)

After obtaining the recombinant adenovirus in HEK293T cells, it was purified using the Vivapure AdenoPACK 20 kit (VS-AVPQ022; Sartorius, Madrid, Spain). Subsequently, the virus titer was determined by plaque-forming assay.
The functionality of the expressed VDR was assessed in human upcyte hepatocytes and HepG2 by measuring the expression of CYP24A1, a well-characterized VDR target gene. Results in Supplementary Figure S1 show that CYP24A1 was highly induced when transfected VDR was activated. The large fold-increase observed is consequence of the very low basal mRNA level of CYP24A1, which is almost null in the absence of active VDR signaling.

HEK 293T cells were transfected with the expression clone and grown until confluence. The cells were lysed, and the supernatant was collected for further amplifications. The collected supernatant was purified using Vivapure AdenoPACK 20 kit (Sartorius Stedim Biotech GmbH, Gottingen, Germany; Cat. # VS-AVPQ020).

An adenovirus carrying the luciferase gene driven by circadian promoter/enhancer elements was constructed using the ViraPower Adenoviral Gateway Expression Kit (K4940-00, Thermo Fisher Scientific). Together with the luciferase gene, the transcription-regulatory region of the hBmal1 gene was excised from the previously constructed vector hBmal1 (−3465–+57) -pGL3^{13 (link)} and the insert was subcloned into the pENTR-1A vector. The resulting vector hBmal1 (−1683–+57) -luc-pENTR-1A was used for recombination with the pAd/PL-DEST vector. The resulting pAd/PL-DEST vector was transfected into HEK293A cells to produce an adenovirus vector carrying hBmal1-Luc. The adenovirus was amplified and purified using the Vivapure AdenoPACK20 Kit (VS-AVPQ020, Sartorius), and 1 × 10^11~12 viral particles were then infected to mice by tail vein injection. Successful luciferase expression was confirmed by in vivo imaging 24 h after infection.