identifying efficient chemical-based nucleic acid transfection … · identifying efficient...

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Identifying efficient chemical-based nucleic acid transfection compound for primary neurons and neuronal cell lines Fanny Prémartin, Valérie Moreau-Toussaint, Thibaut Benchimol, Mégane Denu, Maxime Dumont, Guillaume Freund, Perrine Friedel, Yann Philipson, Malik Hellal, Patrick Erbacher Polyplus-transfection®, Bioparc, 850 Boulevard Sébastien Brant, 67401 Illkirch, France 24 h post-transfection 24 h post-transfection 48 h post-transfection Hep G2 Lipofectamine® 3000 Lipofectamine® 2000 MCF- 10A jetMESSENGER® is a trademark of Polyplus-transfection S.A. Lipofectamine® is a trademark of Life Technologies Corporation. www.polyplus-transfection.com Data provided by K. Le Blay, A. Sebillot, B. Demeneix, S. Remaud of MNHN USM501 UMR-CNRS-7221 « Laboratoire Evolution Des Régulations Endocriniennes », Paris, France. Following jetMESSENGER®-mediated mRNA transfection, the neurospheres were stained with DAPI (cell viability, blue), GFP (transfected cells, green) and GFAP (neuronal cells, red). GFP expression was assayed by fluorescence microscopy in different cell types 24h post-transfection of plasmid encoding GFP (pCMV-EGFP) with FP9219. Cell morphologies are maintained and neurites networks fluorescence are clearly visible following transfection with FP9219. Cell types DNA Transfection efficiency with FP9219 Normal Human Astrocytes (NHA) 57.7% Rat Cortex Neurons (RCN) 20.3% Rat Hippocampal Neurons (RHN) 21% DopaNeurons (from IPS differentiation) 3.3% Abstract : Efficient gene expression in neurons is indispensable for the study of neuronal cell biology, such as investigating gene and protein function, cell behavior and/or cell morphology. The need for more physiologically relevant cellular models has become a requirement to further validate studies performed in neuron cell lines that are easier to transfect compared to primary neurons. Primary neurons are fragile and difficult to transfect, and with currently available transfection method either results in low transfection efficiency or low cell viability. Currently, the most efficient methods for exogenous gene delivery into slow to non-dividing neurons are electroporation or viral-based transduction methods. These methods are often associated with side-effects on cellular viability and morphology. Here we describe the screening of a new library of chemical compounds to identify candidates as potent DNA transfection reagents in different primary neurons (such as primary hippocampal or cortical neurons from rat) and neuronal cell lines. Following the optimization of hits-to-lead, we selected the best candidate based on its high transfection efficiency and its ability to maintain excellent cell viability and morphology. Introduction FP9219 development Normal Human Astrocytes (NHA) Rat Cortex Neurons (RCN) Rat Hippocampal Neurons (RHN) DopaNeurons (from IPS) Efficient gene expression in primary neurons and neuronal cells with FP9219 GFP expression was assayed by fluorescence microscopy in various cell lines 24h post-transfection of mRNA encoding GFP (EGFP-mRNA) with jetMESSENGER®. High efficiencies and viabilities are obtained. jetMESSENGER® is an efficient transfection reagent and preserves cell morphologies. Normal Human Astrocytes (NHA) Rat Cortex Neurons (RCN) DopaNeurons (from IPS) Rat Hippocampal Neurons (RHN) Increased gene expression when switching to mRNA transfection using jetMESSENGER® mRNA-EGFP expression in Neurospheres with jetMESSENGER® DopaNeurons from Induced Pluripotent stem Cells (IPS) are very difficult to transfect. mRNA-EGFP transfection with jetMESSENGER® is highly efficient in these cells, without inducing any cell toxicity. This approach emphasizes transfection efficiency in comparison to DNA transfection with Lipofectamine® 2000. Lipofectamine® 2000 / DNA DopaNeurons (from IPS) DopaNeurons (from IPS) Transfection efficiencies were assayed by fluorescence microscopy or flow cytometry analysis 24h post-transfection of EGFP-mRNA (L-7202, Trilink™) with jetMESSENGER™ or pCMV-EGFP with Lipofectamine® 2000. 0 10 20 30 40 50 60 70 80 90 100 jetMESSENGER® / mRNA Lipofectamine™ 2000 / DNA Transfection efficiency (%) jetMESSENGER® (mRNA) vs. Lipofectamine® 2000 (DNA) – Transfection efficiency RCN DopaNeurons (from IPS) RHN NHA Astrocytes jetMESSENGER® transfection efficiency versus competitor DNA transfection reagent Transfection with FP9219 and jetMESSENGER® preserves cell viability and morphology of sensitive cells as it requires low amount of nucleic acid and volume of reagent, while reaching high transfection efficiency in physiological conditions. Transfection using these reagent is straightforward and provides reproducible results. Approaches : Two different solutions to transfect Neurons or Neuronal cells (DNA or mRNA) Highly efficient: Reach high gene expression in several cells types Cost-effective: Use low reagent volume and DNA/mRNA amounts Biologically relevant: Keep a high cell viability & preserve morphology Simplicity : Transfect with an optimized easy and fast protocol For any questions contact : [email protected] Conclusion Transfection of primary neurons and neuronal cells is a challenge for many researchers. The available DNA transfection reagents usually results in low transfection efficiency and are toxic for the cells. Taking into consideration the difficulties encountered and based on our expertise in transfection, we are currently developing a novel DNA transfection reagent, FP9219. This compound is promising, as it improves DNA delivery and intracellular transport, leading to an efficient gene expression. As an alternative, mRNA transfection using jetMESSENGER® can be an efficient approach to transfect primary neurons and neuronal cells. This poster will present the results obtained with both approaches. A screening of a proprietary chemical compounds library was performed to select new molecules leading to superior DNA transfection efficiency. After hits identification and validation, we optimized their chemistry through structure/activity relationship (SAR) studies to select the best one, FP9219. The easy and fast transfection protocol is an advantage for this ready-to-use compound. FP2919 FP2919 Identification of compounds mediating efficient transfection Hits validation on expanded cell lines Structure optimization Hits-to-lead screening Activity optimization Final reagent jetMESSENGER® / mRNA

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Page 1: Identifying efficient chemical-based nucleic acid transfection … · Identifying efficient chemical-based nucleic acid transfection compound for primary neurons and neuronal cell

Identifying efficient chemical-based nucleic acid transfection compound for primary neurons and neuronal cell lines

Fanny Prémartin, Valérie Moreau-Toussaint, Thibaut Benchimol, Mégane Denu, Maxime Dumont, Guillaume Freund, Perrine Friedel, Yann Philipson, Malik Hellal, Patrick Erbacher

Polyplus-transfection®, Bioparc, 850 Boulevard Sébastien Brant, 67401 Illkirch, France

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

Hep G2

Lipofectamine® 3000

Lipofectamine® 2000

MCF-10A

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

www.polyplus-transfection.com

Data provided by K. Le Blay, A. Sebillot, B. Demeneix, S. Remaud of MNHN USM501 UMR-CNRS-7221« Laboratoire Evolution Des Régulations Endocriniennes », Paris, France.

Following jetMESSENGER®-mediated mRNA transfection, the neurospheres werestained with DAPI (cell viability, blue), GFP (transfected cells, green) and GFAP(neuronal cells, red).

GFP expression was assayed by fluorescence microscopy in different cell types 24hpost-transfection of plasmid encoding GFP (pCMV-EGFP) with FP9219. Cellmorphologies are maintained and neurites networks fluorescence are clearly visiblefollowing transfection with FP9219.

Cell typesDNA Transfection efficiency with

FP9219

Normal Human Astrocytes (NHA) 57.7%

Rat Cortex Neurons (RCN) 20.3%

Rat Hippocampal Neurons (RHN) 21%

DopaNeurons (from IPS differentiation) 3.3%

Abstract : Efficient gene expression in neurons is indispensable for the study of neuronal cell biology, such as investigating gene and protein function, cell behavior and/or cell morphology. The need for more physiologically relevant cellular models has become a requirement to further validate studies performed in neuron cell lines that are easier to transfect compared toprimary neurons. Primary neurons are fragile and difficult to transfect, and with currently available transfection method either results in low transfection efficiency or low cell viability. Currently, the most efficient methods for exogenous gene delivery into slow to non-dividing neurons are electroporation or viral-based transduction methods. These methods are oftenassociated with side-effects on cellular viability and morphology. Here we describe the screening of a new library of chemical compounds to identify candidates as potent DNA transfection reagents in different primary neurons (such as primary hippocampal or cortical neurons from rat) and neuronal cell lines. Following the optimization of hits-to-lead, we selected the bestcandidate based on its high transfection efficiency and its ability to maintain excellent cell viability and morphology.

Introduction

FP9219 development

Normal Human Astrocytes (NHA)

Rat Cortex Neurons (RCN)

Rat Hippocampal Neurons (RHN)

DopaNeurons (from IPS)

Efficient gene expression in primary neurons and neuronal cells with FP9219

GFP expression was assayed by fluorescence microscopy in various cell lines 24h post-transfection of mRNA encoding GFP (EGFP-mRNA) with jetMESSENGER®.High efficiencies and viabilities are obtained. jetMESSENGER® is an efficient transfection reagent and preserves cell morphologies.

Normal Human Astrocytes (NHA)

Rat Cortex Neurons (RCN)

DopaNeurons (from IPS)

Rat Hippocampal Neurons (RHN)

Increased gene expression when switching to mRNA transfection using jetMESSENGER®

mRNA-EGFP expression in Neurospheres with jetMESSENGER®

DopaNeurons from InducedPluripotent stem Cells (IPS)are very difficult to transfect.mRNA-EGFP transfection withjetMESSENGER® is highlyefficient in these cells,without inducing any celltoxicity. This approachemphasizes transfectionefficiency in comparison toDNA transfection withLipofectamine® 2000.

Lipofectamine® 2000 / DNA

DopaNeurons (from IPS)

DopaNeurons (from IPS)

Transfection efficiencies were assayed by fluorescence microscopy or flow cytometry analysis24h post-transfection of EGFP-mRNA (L-7202, Trilink™) with jetMESSENGER™ or pCMV-EGFPwith Lipofectamine® 2000.

0

10

20

30

40

50

60

70

80

90

100

jetMESSENGER® / mRNA Lipofectamine™ 2000 / DNA

Tran

sfe

ctio

n e

ffic

ien

cy(%

)

jetMESSENGER® (mRNA) vs. Lipofectamine® 2000 (DNA) – Transfection efficiency

RCN DopaNeurons (from IPS) RHN NHA Astrocytes

jetMESSENGER® transfection efficiency versus competitor DNA transfection reagent

Transfection with FP9219 and jetMESSENGER® preserves cell viability and morphology ofsensitive cells as it requires low amount of nucleic acid and volume of reagent, whilereaching high transfection efficiency in physiological conditions. Transfection using thesereagent is straightforward and provides reproducible results.

Approaches : Two different solutions to transfect Neurons or Neuronal cells (DNA

or mRNA)

Highly efficient: Reach high gene expression in several cells types

Cost-effective: Use low reagent volume and DNA/mRNA amounts

Biologically relevant: Keep a high cell viability & preserve morphology

Simplicity : Transfect with an optimized easy and fast protocol

For any questions contact : [email protected]

Conclusion

Transfection of primary neurons and neuronal cells is a challenge for many researchers. Theavailable DNA transfection reagents usually results in low transfection efficiency and are toxic forthe cells. Taking into consideration the difficulties encountered and based on our expertise intransfection, we are currently developing a novel DNA transfection reagent, FP9219. This compoundis promising, as it improves DNA delivery and intracellular transport, leading to an efficient geneexpression.As an alternative, mRNA transfection using jetMESSENGER® can be an efficient approach totransfect primary neurons and neuronal cells. This poster will present the results obtained with bothapproaches.

A screening of a proprietary chemical compounds library was performed to select new moleculesleading to superior DNA transfection efficiency. After hits identification and validation, we optimizedtheir chemistry through structure/activity relationship (SAR) studies to select the best one, FP9219.The easy and fast transfection protocol is an advantage for this ready-to-use compound.

FP2919

FP2919

Identification of compounds mediating efficient transfection

Hits validation on expanded cell lines

Structure optimization

Hits-to-lead screening

Activity optimization

Final reagent

jetMESSENGER® / mRNA