156. Delivery of Plasmid DNA to the Nucleus Using Non-Viral Gene Transfection in Tumor Cell Lines and Primary Tumors

Abstract

NovaFECTION (Venn Nova), a standard cationic lipid based transfection method, and Nucleofection (Amaxa), a novel electroporation method, were used to transfect three tumor cell lines (human osteosarcoma U2OS, mouse aggressive neuroblastoma AGN2a, and mouse squamous cell carcinoma SCCVII) and primary AGN2a tumors with a plasmid-based red fluorescent protein (RFP) expression vector. RFP expression was detected by flow cytometry, cell division by CFDA SE staining, and subcellular localization of plasmid by quantitative PCR of the plasmid-encoded neo gene. RFP expression was detected in at least 10% of Nucleofected U2OS cells (0.5 mcg of RFP plasmid or 1011 plasmids /106 cells) within 4 to 6 hours. RFP expression peaked at 12 hours post-Nucleofection. In contrast, RFP expression in NovaFECTED U2OS cells was not detected at 6 hours. NovaFECTED RFP was initially detected in at least 10% of the U2OS cells at 12 hours and peaked at 24 hours. To test the ability to transfect primary AGN2a tumors using these methods, tumors were excised from mice, subjected to collagenase D digestion and viable cells were isolated by density gradient centrifugation. The RFP expression pattern in the transfected primary tumors paralleled that seen with the cultured tumor cell lines: RFP expression began at 5 hours and peaked at 48 hours post-Nucleofection, while NovaFECTION required 24 hours to be initially detected and peaked at 4 days. To determine the relationship between cell division and expression of transfected DNA, three cell lines were CFDA SE stained and Nucleofected with 0.25 mcg of RFP plasmid/106 cells. Rates of cell division differed greatly as Nucleofected U2OS cells divided approximately every 2 hours while AGN2a and SCCVII cells divided only once or twice per 24 hours. For Nucleofected U2OS cells, detection of RFP required 6 hours, and AGN2a and SCCVII cells required 24 hours for detectable expression levels of RFP to be seen. To assess the subcellular localization of plasmid transfected either by Nucleofection or NovaFECTION, the neo gene was amplified by real-time PCR from purified nuclei and compared to total cellular levels. Prior to DNA extraction, cells or nuclei were treated with DNase I to eliminate non-transfected plasmid. In U2OS and SCCVII, greater than 1000 copies of plasmid were detected in the nucleus immediately following Nucleofection. By NovaFECTION, plasmid entry into the nucleus required a minimum of 6 hours, and net plasmid amounts were greater. Therefore, early expression of transfected RFP requires immediate nuclear localization, as demonstrated by Nucleofection. However, nuclear localization does not appear to be sufficient for RFP expression in SCCVII cells. Both U2OS and SCCVII had RFP-vector present in the nucleus at time zero, but only the rapidly dividing U2OS cells demonstrated RFP fluorescence at 6 hours post-Nucleofection. The lack of RFP expression prior to cell division may reflect an as yet undescribed contribution by cell division to plasmid-based gene vector expression.