Construction and Cell Transfection

The CDS region of bovine TTR gene (GenBank Accession Number: {"type":"entrez-nucleotide","attrs":{"text":"NM_173967.3","term_id":"451958145","term_text":"NM_173967.3"}}NM_173967.3) was cloned from adipocytes using the forward primer: 5′-CGGGGTACCATGGCTTCCTTCCGTCTGTTCC-3′ and the reverse primer: 5′-GCTCTAGATCACGCCTTGGGACTGCTGA-3′, then recombined into the pcDNA3.1 (+) plasmid vector between the KpnI and XbaI (TaKaRa, Dalian, China) restriction sites to construct the overexpression vector of bovine TTR gene (OV-TTR). The empty pcDNA3.1 (+) plasmid without any insertion fragment was set as negative control (OV-NC). The plasmids were transfected into cells using Lipofectamine 2000 (Invitrogen, United States) according to the manual. The cells were seeded into 12-well plates in triplicate and transfected with OV-TTR, OV-NC on 7 day after adipogenic induction, respectively. On 9 day and 11 day post-adipogenic induction, the cells were collected for RNA and protein extraction using RNAplus (Takara, Japan) and radio immunoprecipitation assay (RIPA) with 1 mM phenylmethanesulfonyl fluoride (PMSF) (Solarbio, China), respectively.