Construction of apelin lentiviral expression plasmid and preparation of lentiviral particles

GV208-apelin (Genechem, China) was used for subclonning of apelin gene to the lentivector pReceiver-Lv203 (Genecopoeia, Rockville, MD, USA) with CMV promotor, enhanced green fluorescent protein (eGFP) and selection marker puromycin. Primers for amplifying the cDNA of the apelin gene (forward 5′-GCAGGCTTGGAAGGAGTTCGAACCATGAATCTGCGGCTCTGCGTGCAGGC-3′, reverse 5′-CATCGTCTTTGTAGTCCATTACTCTCAGAAAGGCATGGGTCCCTTATGGG-3′) were synthesized from Sangon (Shanghai, China). The pReceiver-Lv203-apelin clone was verified by DNA sequencing (GENEWIZ, China). Lentiviral particles with pReceiver-Lv203-apelin and pReceiver-Lv203 were produced through transfection of HEK293T packaging cells with 3rd generation plasmids system [26]. Lentiviral particles were harvested by ultrafiltration with 100 kDa spin column (Merck, Germany), dispensed in aliquots, and stored at − 80 °C until use. Transfection efficiency was examined by EGFP under a fluorescence microscope (Olympus, Japan), and viral titer was determined according to the equation: virus titer (pfu/mL) = cell number in each well × virus dilution factor × 10/volume of added virus fluid (mL).