Improved gene expression in hard-to-transfect cells with jetOPTIMUS® Transfection ReagentGuillaume Freund, Valérie Moreau-Toussaint, Mégane Denu, Thibaut Benchimol, Fanny Prémartin, Mathieu Porte, Maxime Dumont, Yann Philipson, Fabrice Stock, Malik Hellal, Patrick ErbacherPolyplus-transfection®, Bioparc, 850 Boulevard Sébastien Brant, 67401 Illkirch, France

Superior gene expression in hard-to-transfect cells

24 h post-transfection24 h post-transfection 48 h post-transfection

Abstract High EGFP expression in various cell types

jetOPTIMUS® development

Maintained gene expression post-transfection

Excellent cell viability and high gene expression using low DNA amount

Transfection efficiency was assessed by FACS analysis 24 h after transfection of plasmid DNA encoding forEGFP (pCMV-EGFP) with jetOPTIMUS®, Lipofectamine® 2000 and Lipofectamine® 3000 in primary cells andcancer cell lines.

GFP expression was assayed by fluorescencemicroscopy in Vero cells 24 h after transfection.

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96-well 24-well 6-well T-25 T-75

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Transfection efficiency was assessed by FACS analysis in HeLa cells 24 h after transfection of EGFPplasmid (pCMV-EGFP) with jetOPTIMUS® .

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Cell viability (in blue) was assessed with XTT analysis in HeLa cells 24 h after transfection of EGFP plasmid(pCMV-EGFP) with jetOPTIMUS® in 24-well plates. GFP expression (in green) was analyzed by FACS also 24h after transfection.

HUVEC

GFP expression was assayed by fluorescence microscopy in various cell lines 24 h after transfection withjetOPTIMUS®.

Hep G2

Human primary fibroblasts

jetOPTIMUS®

Lipofectamine® 3000

Lipofectamine® 2000

Neuro2a

GFP expression was assessed by FACS in HEK-293, Vero and Hep G2 cells 24 h and 48 h after transfection ofplasmid DNA encoding for EGFP (pCMV-EGFP) with jetOPTIMUS®.

MCF-10A

jetOPTIMUS® is a trademark of Polyplus-transfection S.A.Lipofectamine® is a trademark of Life Technologies Corporation.

FuGENE® is a trademark of Fugent, LLC., USA.

Transfection with jetOPTIMUS® preserves cell viability and morphology of sensitive cells as it requires lowestamount of DNA and volume of reagent while reaching high transfection efficiency in physiologicalconditions. This reagent associates higher transfection efficiency and lower toxicity compared to othercommercially available delivery solutions. DNA transfection using jetOPTIMUS® is straightforward andprovides highly reproducible results.

Highly efficient: Reach maximal gene expression in many cell types

Cost-effective: Use lowest reagent volume and DNA quantity

Biologically relevant: Keep an excellent cell viability & morphology

Time-saving: Transfect with an optimized ready-to-use protocol

Best-in-class DNA transfection reagent

HEK293-F CHO-S

DNA transfection remains a limiting factor for many researchers working with primary cells and specificcell lines. Considering the limiting steps in these hard-to-transfect cell types and based on our knowledgeand expertise in transfection, we have addressed this issue by developing a novel DNA transfectionreagent, jetOPTIMUS®. This reagent improves DNA delivery and intracellular transport ending by highergene expression in hard-to-transfect primary cells and cell lines. This reagent associates highertransfection efficiency and lower toxicity compared to the best commercially available competitors. DNAtransfection reagent jetOPTIMUS® is easy to use and provides highly reproducible results.

MDCK

mHippoE-14 mHypoA-2/12

GFP expression was assayed by fluorescence microscopy inSH-SY5Y (neuroblastoma cell line) 24 h or 48 h aftertransfection with jetOPTIMUS®.

Data kindly provided by Oya ARI UYAR (Gebze Technical University,Kocaeli, Turkey.)

24 h post-transfection 48 h post-transfection

Data kindly provided by Oya ARI UYAR (Gebze Technical University, Kocaeli, Turkey).

GFP expression was assayed by fluorescence microscopy in mHippoE14 (Embryonic Mouse Hippocampal cellline) and mHypoA-2/12 (Adult Mouse Hypothalamus cell line ) 24 h after transfection with jetOPTIMUS ®

GFP expression was assayed by fluorescence microscopy inHeLa cells 24 h after transfection with jetOPTIMUS®.

Final reagent

Activity optimization

Hits-to-lead screening

Structure optimization

Hits validation on expanded cell lines

Identification of compounds mediating efficient transfection

High-throughput screening of a proprietary chemical compounds library was performed to select newmolecules leading to superior transfection efficiency while maintaining excellent cell viability in differentcell types. After hits identification and validation, we optimized their chemistry through structure/activityrelationship (SAR) studies to select the best one, jetOPTIMUS®.

Easy to scale up and down

DNA delivery Synthetic vector

Difficult to transfect cells Low DNA and minor toxicity

jetOPTIMUS®

The jetOPTIMUS® project

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www.polyplus-transfection.com

250 ng DNA 500 ng DNA

250 ng DNA 500 ng DNA

Cell viability and morphology was assayed in HeLa cells bymicroscopy 24 h after transfection with jetOPTIMUS®.

Human mesenchymal stem cells

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HEK-293 Vero Hep G2

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24h 48h

Optimal conditions

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HeLa MCF-10A Hep G2 Vero HUVEC hMSC CPRE-2

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jetOPTIMUS® Lipofectamine® 3000 Lipofectamine® 2000

Cancer cell lines Primary and stem cells