The introduction of transgenes into stem cells has shown to be a valuable experimental technique for studying stem cell biology. Transfecting stem cells without inhibiting cell viability and cell growth has shown to be difficult. DNA-In® Stem Transfection Reagent offers a simple, robust and reproducible method for delivering DNA into a wide range of stem cells, including neural stem cells. Formulated and optimized specifically for embryonic and adult stem cells, DNA-In® Stem is a new-generation transfection reagent that enables high efficiency transfection while maintaining maximum cell viability and cell growth.

In this post, I invite you to discover the benefits of using DNA-In® Stem Transfection Reagent vs. other reagents. A lot of pictures and graphs rather than long descriptions! Last but not least, DNA-In® Stem Transfection Reagent is less expensive compared to Lipofectamine reagents…

High efficiency CRISPR-Cas9 Delivery

Figure 1. Neural progenitor cells (HT223-B) from adult brain were transfected with pEF1a-Cas9-2A-GFP plasmid (Addgene) using DNA-In® Stem (left) and Lipofectamine 3000 (right) . Data courtesy of Sasha Beylina, Staff Scientist, NIH/NIA
Figure 1. Neural progenitor cells (HT223-B) from adult brain were transfected with pEF1a-Cas9-2A-GFP plasmid (Addgene) using DNA-In® Stem (left) and Lipofectamine 3000 (right) . Data courtesy of Sasha Beylina, Staff Scientist, NIH/NIA

High efficiency, low cytotoxicity

Figure 2. Data above show cells transfected with DNA-In® Stem Transfection Reagent. Cells were plated in 24-well plates to give 60-70% confluency on the day of transfection. Cells were transfected with various amounts of a DNA plasmid, containing eGFP behind an EF1-alpha promoter using 1µl DNA-In® Stem. Cells were analyzed 24-hours and 48-hours (not shown) after transfection. Above, representative images of normal human induced pluripotent stem cells (iPSC)-derived neural stem cells (NSC), Human embryonic stem cells, adipose-derived mesenchymal stem cells (MSC) and Huntington's Disease (HD)-specific iPSCs show consistently high efficiency with low toxicity when using DNA-In® Stem transfection reagent.

Figure 2. Data above show cells transfected with DNA-In® Stem Transfection Reagent. Cells were plated in 24-well plates to give 60-70% confluency on the day of transfection. Cells were transfected with various amounts of a DNA plasmid, containing eGFP behind an EF1-alpha promoter using 1µl DNA-In® Stem. Cells were analyzed 24-hours and 48-hours (not shown) after transfection. Above, representative images of normal human induced pluripotent stem cells (iPSC)-derived neural stem cells (NSC), Human embryonic stem cells, adipose-derived mesenchymal stem cells (MSC) and Huntington’s Disease (HD)-specific iPSCs show consistently high efficiency with low toxicity when using DNA-In® Stem transfection reagent.

Outperforms competitors across a range of cell types

DNA-In_Stem_vs_Competitor-NSC1DNA-In_Stem_vs_Competitor-MSC2DNA-In_Stem_vs_Competitor-hESC3DNA-In_Stem_vs_Competitor-HD4
Figure 3 (a-d). DNA-In® Stem requires up to 4X less DNA vs competitor reagents for maximum performance. Human iPSC-derived neural stem cells (NSC), Human embryonic stem cells, adipose-derived Mesencymal Stem Cells and Huntington’s Disease (HD)-specific iPSCs plated in 24-well plates were transfected with DNA-In® Stem, Lipofectamine®2000 (L2000) and 3000 (L3000) using various amounts (µl) of reagent. Cells transfected with DNA-In® Stem were transfected with both low and high amounts of DNA. Cells were also transfected with L2000 and L3000 using their recommended 500ng of DNA.

Consistent, robust performance

robust 1 robust 2 robust 3 robust 4Figure 4 (a-d). DNA-In® Stem shows robust performance across a range of DNA amounts in stem cells to product high transfection efficiency. Stem cells, including plated in 24-well plates were transfected with various amounts of DNA-In® Stem and DNA. Cells were then observed 24-hours (above data) and 48-hours (not shown) post-transfection. Above data for Human iPSC-derived neural stem cells (NSC), Human embryonic stem cells, adipose-derived Mesencymal Stem Cells and Huntington’s Disease (HD)-specific iPSCs show consistently high efficiency with low toxicity when using DNA-In® Stem Transfection Reagent.

Protocol

DNA-In Stem quick startDNA-In® Stem Reagent is a formulation of chemically defined compounds, and is completely free of animal-derived components. The protocol provided has been optimized to achieve the highest number of cells transfected in a population (%CT), without toxicity. Higher expression levels can be obtained later by addition of more DNA if required. It is recommended that the first set of experiments be done using a GFP reporter system to optimize percent cells transfected with DNA-In® Stem Reagent. The amount of plasmid DNA/DNA-In® Stem Reagent complex that is added to cells is a critical factor in determining percent cells transfected, level of expression, and cellular toxicity. This reagent has been optimized for intracellular delivery of DNA into Stem cells in the presence of serum or low protein medium at a cell density of 60% to 80%. High levels of expression can be achieved using the amount of DNA-In® Stem Reagent and DNA amounts recommended in the following protocol. For best results, it is important to empirically determine the optimal amount of DNA and DNA-In® Stem Reagent for any given cell type. If toxicity is observed, reducing the amount of DNA may reduce toxicity while still maintaining high levels of expression and % cell transfected.

Download your protocol copy here! 222_DNA-In_Stem_Protocol

mRNA transfection?

mRNA-In® Stem,  is a new mRNA transfection reagent specifically designed and optimized for high efficiency mRNA delivery with exceptionally low cytotoxicity in stem cells and  hard-to-transfect primary cells. mRNA-In® Stem requires very low amounts of RNA to achieve maximum delivery while maintaining optimal cell viability.

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DNA-In® is a Molecular Transfer, Inc trademark
Lipofectamine® is a Life Technologies, Inc registered trademark

Written by Jean-François Têtu, PhD
Jean-François Têtu is Sales Manager at tebu-bio for the south of Europe and also for the UK and Eire. He also fervently defends and promotes anything from his home territory, the north of France.