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Optimizing the process of nucleofection for professional antigen presenting cells.

Mullins CS, Wegner T, Klar E, Classen CF, Linnebacher M - BMC Res Notes (2015)

Bottom Line: In this study, we compared B cells to DC with regard to nucleofection efficiency and intensity of resulting antigen expression.And no differences with regard to nucleofectability were observed between the two cell types.Using IVT mRNA omits the danger of genomic integration and plasmid DNA constructs permit a more potent and longer lasting antigen expression.

View Article: PubMed Central - PubMed

Affiliation: Molecular Oncology and Immunotherapy, Department of General Surgery, University Hospital Rostock, Schillingallee 35, 18057, Rostock, Germany. christina.mullins@uni-rostock.de.

ABSTRACT

Background: In times of rapidly increasing numbers of immunological approaches entering the clinics, antigen delivery becomes a pivotal process. The genuine way of rendering antigen presenting cells (APC) antigen specific, largely influences the outcome of the immune response. Short peptides bear the demerit of HLA restriction, whereas the proper way of delivery for long peptide sequences is currently a matter of debate. Electroporation is a reliable method for antigen delivery, especially using nucleic acids. The nucleofection process is based on this approach with the twist of further ensuring delivery also into the nucleus. Beside the form of antigen, the type of APC used for immune response induction may be crucial. Dendritic cells (DC) are by far the most commonly used APC; however B cells have entered this field as well and have gained wide acceptance.

Results: In this study, we compared B cells to DC with regard to nucleofection efficiency and intensity of resulting antigen expression. APC were transfected either with plasmid DNA containing the reporter gene green fluorescent protein (GFP) or directly with in vitro-transcribed (IVT) GPF mRNA as a surrogate antigen. Out of nearly 100 different nucleofection programs tested, the top five for each cell type were identified and validated using cells from cancer patients. Flow cytometric analyses of transfected cells determining GFP expression and viability revealed a reverse correlation of efficiency and viability. Finally, donor dependant variances were analyzed.

Conclusion: In summary, nucleofection of both DC and B cells is feasible with plasmid DNA and IVT mRNA. And no differences with regard to nucleofectability were observed between the two cell types. Using IVT mRNA omits the danger of genomic integration and plasmid DNA constructs permit a more potent and longer lasting antigen expression.

No MeSH data available.


Related in: MedlinePlus

Comparison of B cell nucleofection with plasmid DNA and IVT mRNA. The dot plots for flow cytometric analyses of a cancer patient’s B cells post nucleofection with either 1 µg plasmid DNA, 10 µg IVT mRNA or without any nucleic acid (serving as reference) are depicted
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Fig8: Comparison of B cell nucleofection with plasmid DNA and IVT mRNA. The dot plots for flow cytometric analyses of a cancer patient’s B cells post nucleofection with either 1 µg plasmid DNA, 10 µg IVT mRNA or without any nucleic acid (serving as reference) are depicted

Mentions: The amount of nucleic acid necessary for highly successful nucleofection is tenfold higher for IVT mRNA compared to plasmid DNA (Fig. 7). With regard to the intensity of protein expression—as determined by fluorescence intensity in flow cytometry—plasmid DNA is more potent than IVT mRNA (Fig. 8). Yet, for IVT mRNA an increase in efficacy could be achieved by using more RNA (3 vs. 10 µg); this was not the case for plasmid DNA, where rather a decrease in viability was observed (data not shown). Finally, in terms of viability, no great differences were observed between the two types of nucleic acids (Additional file 4: Figure S2, Additional file 5: Figure S3).Fig. 8


Optimizing the process of nucleofection for professional antigen presenting cells.

Mullins CS, Wegner T, Klar E, Classen CF, Linnebacher M - BMC Res Notes (2015)

Comparison of B cell nucleofection with plasmid DNA and IVT mRNA. The dot plots for flow cytometric analyses of a cancer patient’s B cells post nucleofection with either 1 µg plasmid DNA, 10 µg IVT mRNA or without any nucleic acid (serving as reference) are depicted
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4581479&req=5

Fig8: Comparison of B cell nucleofection with plasmid DNA and IVT mRNA. The dot plots for flow cytometric analyses of a cancer patient’s B cells post nucleofection with either 1 µg plasmid DNA, 10 µg IVT mRNA or without any nucleic acid (serving as reference) are depicted
Mentions: The amount of nucleic acid necessary for highly successful nucleofection is tenfold higher for IVT mRNA compared to plasmid DNA (Fig. 7). With regard to the intensity of protein expression—as determined by fluorescence intensity in flow cytometry—plasmid DNA is more potent than IVT mRNA (Fig. 8). Yet, for IVT mRNA an increase in efficacy could be achieved by using more RNA (3 vs. 10 µg); this was not the case for plasmid DNA, where rather a decrease in viability was observed (data not shown). Finally, in terms of viability, no great differences were observed between the two types of nucleic acids (Additional file 4: Figure S2, Additional file 5: Figure S3).Fig. 8

Bottom Line: In this study, we compared B cells to DC with regard to nucleofection efficiency and intensity of resulting antigen expression.And no differences with regard to nucleofectability were observed between the two cell types.Using IVT mRNA omits the danger of genomic integration and plasmid DNA constructs permit a more potent and longer lasting antigen expression.

View Article: PubMed Central - PubMed

Affiliation: Molecular Oncology and Immunotherapy, Department of General Surgery, University Hospital Rostock, Schillingallee 35, 18057, Rostock, Germany. christina.mullins@uni-rostock.de.

ABSTRACT

Background: In times of rapidly increasing numbers of immunological approaches entering the clinics, antigen delivery becomes a pivotal process. The genuine way of rendering antigen presenting cells (APC) antigen specific, largely influences the outcome of the immune response. Short peptides bear the demerit of HLA restriction, whereas the proper way of delivery for long peptide sequences is currently a matter of debate. Electroporation is a reliable method for antigen delivery, especially using nucleic acids. The nucleofection process is based on this approach with the twist of further ensuring delivery also into the nucleus. Beside the form of antigen, the type of APC used for immune response induction may be crucial. Dendritic cells (DC) are by far the most commonly used APC; however B cells have entered this field as well and have gained wide acceptance.

Results: In this study, we compared B cells to DC with regard to nucleofection efficiency and intensity of resulting antigen expression. APC were transfected either with plasmid DNA containing the reporter gene green fluorescent protein (GFP) or directly with in vitro-transcribed (IVT) GPF mRNA as a surrogate antigen. Out of nearly 100 different nucleofection programs tested, the top five for each cell type were identified and validated using cells from cancer patients. Flow cytometric analyses of transfected cells determining GFP expression and viability revealed a reverse correlation of efficiency and viability. Finally, donor dependant variances were analyzed.

Conclusion: In summary, nucleofection of both DC and B cells is feasible with plasmid DNA and IVT mRNA. And no differences with regard to nucleofectability were observed between the two cell types. Using IVT mRNA omits the danger of genomic integration and plasmid DNA constructs permit a more potent and longer lasting antigen expression.

No MeSH data available.


Related in: MedlinePlus