Pgv186 retroviral vector

Retroviruses were used to introduce hCLOCK into the HUVECs and serve as vectors for negative control cells and scrambled control cells.
Stable transfectants overexpressing hCLOCK (GenBank Accession Number: NM_004898) were generated via retroviral transduction using a pGV186 retroviral vector (GeneChem Co., Ltd., Shanghai, China). As a control, a retroviral vector expressing green fluorescent protein alone was also generated.
The short hairpin RNA (shRNA) sequences targeting hCLOCK were constructed using a pGV113 retroviral vector (GeneChem Co., Ltd., Shanghai, China). Scrambled shRNA expressing vectors (SCR) serving as controls were made in a similar fashion.

The full-length complementary (c)DNA of hClock (Genbank accession number, NM_004898) was amplified and inserted into the pcDNA3-Flag vector (Invitrogen Life Technologies, Carlsbad, CA, USA) in order to generate the pcDNA3-Flag-hClock expression plasmid. The cDNA fragment of Flag-hClock was then inserted into pGV186 retroviral vector (Shanghai GeneChem Co., Ltd., Shanghai, China) to generate pGV186-Flag-hClock. All the constructs were confirmed by DNA sequencing, which was performed by Shanghai GeneChem Co., Ltd.