Generation of human WT and Y872F MERTK pMSCV vectors for transduction into Mertk−/− mouse macrophages

Human MERTK (NM_006343.2) was cloned into pMSCV-puro retroviral vector (Addgene) at the Xho I and Eco RI restriction sites (Genewiz). Tyr872 of human MERTK was mutated to phenylalanine using the PCR-based QuikChange mutagenesis system (Stratagene). BOSC23 cells (5 × 106 cells) were transfected with 1 μg of pMSCV-WT Mertk or pMSCV-Y872F MERTK together with 1 μg of pCL-Eco (Addgene) and 2 μg of pMD2.G (Addgene) using 16 ml of LipoD293 transfection reagent (SignaGen). Conditioned medium was collected 48 hours after transfection and filtered through 0.45-μm filters. The medium was then used to transduce BMDMs from Mertk−/− mice.

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