Background

Transfection is one of the most commonly used techniques in molecular biology (Stoll and Calos, 2002; Kim and Eberwine, 2010). Transfection is the process of introducing nucleic acid (DNA that carries a gene of interest or mRNA) into target cells that then eventually express the desired nucleic acid or protein. There are several biological, chemical, and physical methods for introducing nucleic acids into cells (Stoll and Calos, 2002; Kim and Eberwine, 2010; Zhou et al., 2016). However, all these methods are variable and don’t assess the cell transfection efficiency, cell toxicity and the level of gene expression within the same experiment. To truly optimize cellular transfection, a sensitive and robust detection assay is required to quantify and optimize the efficiency of different transfection methods to deliver the target gene into the cytosol and facilitate protein expression, while reducing cell toxicity.

Herein, we demonstrate the development of a flow-cytometric assay to determine transfection efficiency by labeling a reporter plasmid with Label IT^® Tracker^TM (Homann et al., 2017) (Figure 1). This method does not depend on co-transfection of two different plasmids and simultaneously quantifies cell death, uptake of the labeled plasmid during transient transfection, and expression of the target protein. We demonstrate that this method can be used as a tool to i) optimize transfection efficiency in 2 independent standard cell lines, ii) quantify cellular toxicity of different transfection methods, iii) determine uptake of DNA into difficult to transfect cells via electroporation without the need to use co-transfection of GFP plasmid that can further reduce the efficiency of transfection. This flow cytometric method can be directly applied to optimize several transfection methods including gene therapy strategies (e.g., CRISPR/Cas system).


Figure 1. Experimental design for determination of transfection efficiency by flow cytometric method. The plasmid DNA was labeled with FITC by DNA label IT@ tracker. After transfection, cells were detected by flow cytometry. The FITC fluorochrome is used to detect intracellular levels of the transfected plasmid that has been labeled with FITC (Label IT tracker, green). The second fluorochrome is used to quantify expression of the target protein (by directly measuring fluorescence of the expressed protein if the target protein is fluorescent or by using a fluorescent-labeled antibody against the target protein, red). Either Q1+Q2 (DNA signal) or Q2+Q3 (protein signal) should be used as readouts of transfection efficiency.