Pires egfp plasmid

The cDNA of mouse Flt3L was cloned by RT-PCR of total splenic RNA from C57BL/6 mice, and was used to generate a construct encoding soluble FLAG and OLLAS tagged Flt3L, internal ribosomal entry site (IRES), and enhanced green fluorescence protein (EGFP), i.e., SFO.Flt3L-IRES-EGFP. The GenBank accession number for the sequence including the extracellular domain of mouse Flt3L is GU168042, and the IRES-EGFP sequence is from pIRES-EGFP plasmid (Clontech, Mountain View, CA, USA). CHO cells were then transfected using Lipofectamine 2000 (Life Technologies), with a mammalian expression vector plasmid encoding SFO.Flt3L-IRES-EGFP under CMV promoter and a neomycin resistance gene, constructed with the backbone of pEGFP-N1 (Clontech). CHO/Flt3L cells stably expressing the SFO.Flt3L-IRES-EGFP were produced by the following steps: (i) treatment of transfected CHO cells with G418 (1.5 mg/ml) for 1 week; (ii) enrichment of EGFP-positive CHO cells with FACSAria II cell sorter (BD Biosciences, San Diego, CA, USA); (iii) generation of clonal cells by limiting dilutions of FACS-sorted EGFP-high CHO/Flt3L cells; and (iv) selection of CHO/Flt3L clones after evaluating both levels of EGFP expression by FACS analysis and Flt3L secretion by anti-OLLAS Western blot analysis (22 (link)). Chosen CHO/Flt3L cells were cultured in cell culture flasks with DMC7 to produce CHO/Flt3L-conditioned medium.