Transfection of murine dendritic cell line (JAWS II) by a nonviral transfection reagent.
Dendritic cells are the most potent antigen-presenting cells that initiate and modulate the host immune system. Based on their immunostimulatory activity, a variety of strategies have been developed to use dendritic cells as vaccines and immunotherapeutic agents against infection and cancer. Genetically modified dendritic cells are useful for immunotherapeutic purposes because of their sustained activity in vivo. However, transfection of dendritic cells with plasmid DNA has been very difficult. While the viral transfection is associated with nonspecific activation of dendritic cells, commonly used nonviral transfection reagents have a low efficiency of transfection. Here we describe an improved, simple, less time-consuming transfection protocol using the nonviral nonliposomal lipid polymer, TransIT-TKO transfection reagent, for transfecting murine dendritic cells (JAWS II) with the gene that encodes Coccidioides immitis antigen 2 (Ag2). The JAWS II cells were cotransfected with pHYG-enhanced green fluorescent protein (EGFP) and pVR1012-C. immitis Ag2 plasmid DNAs using TransIT-TKO reagent. We reproducibly obtained 30%-50% transfection efficiency. The transfected cells maintained their immature phenotype and were functionally active. In addition, the flexibility of this agent for expressing multiple antigens (GFP and C. immitis Ag2) offers an advantage of delivering multiple immunogens.
D000818:Animals; D002460:Cell Line; D003713:Dendritic Cells / Q000378:metabolism*; D005656:Fungal Proteins; D005786:Gene Expression Regulation; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics*; D007202:Indicators and Reagents; D051379:Mice; D008810:Mice, Inbred C57BL; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics*; D014162:Transfection / Q000379:methods*; D014780:Viruses / Q000235:genetics
D005656:Fungal Proteins; D006023:Glycoproteins; D007202:Indicators and Reagents; D011994:Recombinant Proteins; C102758:antigen 2, Coccidioides