March 1-31, 2018, MTC/Karolinska Institutet, Stockholm, Sweden

Aims

Train delivery of plasmid DNA by “golden standard” method by injection and electroporation compared to chemical transfection, and assess resulting immune response to encoded protein.

Methods

Training used plasmid DNA encoding near-infrared reporter protein iRFP670 (kindly provided by Prof Vald Verkhusha, Albert Einstein University, USA). Plasmid DNA, obtained by cultivation of E coli, was purified from lyzed bacterial cells using Quigene column chromatography. Complexes of DNA with dendrimers were formed using protocols during Riga training 2017. Complexes were injected into BALB/c mice, and near-infrered signal resulting from expression of iRFP670 was assessed by imaging (Spectrum). Control mice received injections of plasmid DNA followed by electroporation. At experimental end-point, splenes were processed to obtain splenocytes. Cellular immune response to iRFP was assessed by IFN-g ELISpot.

Detection of fluorescence in positions of injections of plasmid encoding infrared reporter protein iRFP670 in mice receiving injection of iRFP670 encoding plasmid piRFP670 complexed to Generation IV dendrimer (group 1) versus mice receiving piRFP670 injection followed by electroporation (group 2). Ilze Fridrihzone, Maksim Ionov and Stefan Petkov (unpublished).

Overall results

Trainees obtained theoretical and practical knowledge on DNA delivery using intradermal injection and electroporation, visualization using Caliper IVIS device with quantification techniques using Perkin Elmer Living Image software 4.5.2, and insights into ELISA and ELISpot. In vivo results confirmed observations made in cell lines. Most efficient delivery of plasmid DNA encoding iRFP670 was supported by electroporation, followed by DNA complexed to Generation III dendrimers. DNA complexed to dendrimers of generation IV was was not delivered. Introduction of iRFP670 DNA induced no anti-iRFP67 immune response. This demonstrated the applicability of iRFP670 as a reporter of tumor growth in immuno-competent mice.