Cloning and Mutagenesis of MERTK Promoter

To construct a reporter plasmid containing the MERTK promoter region, this 1,625-bp region in MERTK was amplified from genomic DNA samples, using the NM_006343.2 reference sequence and primers that contained recognition sites for the HindIII and XhoI restriction endonucleases. The amplified products were inserted into the pGL4.11b[luc2] vector (Promega Corporation, Fitchburg, WI, USA). To construct a plasmid containing the wild-type MERTK gene, a vector (Addgene plasmid 23900) was purchased (Addgene, Cambridge, MA, USA)24 (link) and subcloned into the pcDNA3.1 (+) vector (Life Technologies Corporation). Genetic variations in the promoter and in the coding region were obtained using the QuikChange^® II Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA). All DNA sequences were confirmed by direct sequencing. The primers used in this study are listed in Supplementary Table 1.