2.5. Adenovirus Transduction of Intramuscular Preadipocytes

For overexpression analysis, the ACSL4 CDS sequence was cloned into the Y4261 adenovirus plasmid and transfected in HEK293 cells to package the recombinant adenoviral vector containing the ACSL4 gene, according to the manufacturer instructions (Genepharm Co., Ltd., Shanghai, China). The pig intramuscular preadipocyte cells were seeded into 12 well culture plates at 80% confluence and the adenovirus was added to the medium. After 48 h of transfection, the cells were collected and total RNA and protein were extracted for expression analysis. Adenovirus ADV4-ACSL4 and ADV4-NC (both at 1011 pfu/mL) were synthesized by Gene Pharma. The preadipocytes were cultured in a six-well plate, and adenovirus was added (MOI = 50) when the cells reached 80–90% confluence. Fluorescence expression was observed after 48 h. The cells were collected 72 h later for further analysis.