Immortalized Chicken Preadipocyte Culture and Cloning

A cell line of immortalized chicken preadipocytes (ICP1)^{88 (link)} was a kind gift of the Poultry Breeding Group of the College of Animal Science and Technology, Northeast Agricultural University, China, and was cultured in DMEM/F12 cell culture medium with 10% FBS, at 37 °C with 5% CO₂. ICP1 preadipocytes were seeded in 6-well plates for further transfection using Lipofectamine 2000 (Invitrogen), and the transfection procedure was performed according to the manufacturer’s instructions. After a 48 h recovery period, the cells were supplemented with 3 μg/mL of puromycin (Sigma-Aldrich, MO, USA) to screen out cells which have not been successfully transfected into the plasmids (ie, negative cells). Once the cell clone is formed, cells were harvested using 0.25% trypsin/EDTA (Gibco, Gaithersburg, MD, USA), and the cell density was calculated using a handheld automated cell counter (Millipore, Darmstadt, Germany). Single cells were plated in each well of a 96-well plate by limiting dilution and then cultured for 10 d in the cell culture medium. The medium was replaced every 4 d. Confluent cell colonies were propagated and genotyped by PCR and sequencing. Primer sets used for PCR are listed in Supplementary Table 19.