FAQ: DNA transfection

jetOPTIMUS® is our recommended reagent for day-to-day transfection of adherent cells, even for hard-to-transfect cells. It is well suitable for co-delivery of several plasmids and small-scale virus production. Transfection with jetOPTIMUS® leads to higher transfection efficiencies and gene expression with minimal impact on cell viability and morphology.

jetPRIME® is a polyvalent transfection reagent that ensures effective and reproducible DNA and siRNA transfection in several adherent cell lines. This versatile reagent is also perfect for co-transfection of DNA and siRNA. Transfection with jetPRIME® requires only low amounts of nucleic acid, hence resulting in very low cytotoxicity.

jetPEI® transfection reagent is particularly well suited for automated or manual HTS (High Throughput Screening) with three protocols available: reverse, batch and forward. jetPEI® provides highly reproducible results and the DNA/jetPEI® complexes are stable for up to 4 hours.

For co-transfection of multiple plasmids, we recommend that the total DNA amount per well does not exceed the maximal DNA amount indicated in the jetOPTIMUS® protocol. The ratio to use for each plasmid depends on the size of the plasmid, the plasmid construct and the expected expression level of each plasmid. Each plasmid should represent at least 10% of the total DNA amount per well.

For plasmid DNA/siRNA co-transfection, we recommend jetPRIME® reagent. Please refer to the standard protocol for DNA and siRNA co-transfection available on the jetPRIME page: https://www.polyplus-transfection.com/products/jetprime/

For DNA/ssODN co-transfection (single-stranded oligodeoxynucleotides), the same protocol may be applied.

jetOPTIMUS® is well suitable for DNA transfection of hard-to transfect cells such as primary cells (epithelial, hepatocyte, endothelial, fibroblast, etc…), stem cells (embryonic, mesenchymal, induced pluripotent) and cancer cell lines.

However, some suspension and primary cells, such as lymphocyte cell lines, fibroblasts or neurons, are very difficult to transfect with DNA Polyplus® developed a lipid-based transfection reagent jetMESSENGER®. It was designed for mRNA delivery and showed high efficiency on a wide variety of difficult to transfect cells including lymphoma and blood cell lines, neurons and various primary cells. Compared to DNA, mRNA transfection leads to a high percentage of transfected cells, faster protein expression following transfection, and no risk of genomic insertion in contrast to plasmid or viral vectors

For optimal transfection efficiency using jetOPTIMUS® or jetPRIME®, we recommend not exceeding 10 minutes of complexation. Hence, for applications requiring longer incubation times such as High Throughput Screening, we recommend using jetPEI®, as jetPEI®/DNA and DNA/jetOPTIMUS complexes are stable for up to 4 hours.

There is no size restriction for DNA transfection using those reagents, However, it should be pointed out that for the same amount of DNA, the gene copy number will be lower for large DNA constructs than for smaller plasmids.

jetOPTIMUS® interacts with DNA, forming positively charged complexes which penetrate into the cell through endocytosis. Endosomes release DNA in the cytosol via the proton sponge mechanism (Behr, Chimia 1997). The plasmid mostly reaches the nucleus during mitosis, when nuclear envelope disappears. For this reason, primary cells or cell lines with slow cell division rates are usually hard-to-transfect.

For more information about transfection mechanisms, please consult: https://www.polyplus-transfection.com/technologies/what-is-transfection/