Peg it virus precipitation solution

Lentiviral overexpression of Prdm16 was performed in primary proliferating preadipocytes derived from iWAT followed by Ctcflos KD at the first day of differentiation. FLAG‐Prdm16 open reading frame was PCR‐amplified from MSCV‐Prdm16 (gift from Bruce Spiegelman (Addgene plasmid # 15504; http://n2t.net/addgene:15504; RRID:Addgene_15504) (Seale et al,2007)) and cloned into pCDH‐PGK. pCDH‐PGK‐FLAG‐Prdm16 or pCDH‐PGK‐tGFP, as control, were co‐transfected together with second‐generation packaging plasmids psPAX2 and pMD2.G into HEK 293T cells using PolyFect transfection reagent (Qiagen, # 301105) for production of lentiviral particles. Medium supernatant containing viral particles was collected at three consecutive days and concentrated using PEG‐it Virus Precipitation Solution (Biocat, # LV810A‐1‐SBI). Physical virus titer was determined by One‐Wash Lentivirus Titer Kit, HIV‐1 p24 ELISA (Origene, #TR30038), and primary iWAT preadipocytes were transduced at 50% confluency with multiplicity of infection (MOI) 500 or 800 using polybrene (8 µg/ml) for 24 h. At 80% confluency, transduced preadipocytes were induced for 2 days and ASO‐mediated Ctcflos KD was performed at the first day of differentiation as described above. Cells were harvested 72 h after transfection and analyzed for gene expression by qRT–PCR.

Lentiviral plasmid pLenti-MP2 was a gift from Pantelis Tsoulfas^{63 (link)} and was modified by inserting IRES RFP cassette via XbaI and EcoRV restriction sites. Human GPR180 coding sequence (GenScript) was cloned under CMV promoter using XhoI and XbaI restriction sites using standard cloning techniques. GPR180 and control RFP lentiviruses were generated using pMDG2 and PAX2 packaging vectors in HEK-293T cells. Viral particles were precipitated by PEG-it Virus precipitation solution (BioCat). Biological titer was determined based on RFP positive HEK-293T cells transduced with virus. Mature hMADS-derived adipocytes were infected with 2 MOI with the aid of polybrene at final concentration of 8 µg/ml and Opti-MEM I reduced serum (Gibco).