sh-sy5y: transfection of sirna · sh-sy5y: transfection of sirna ... lipocalyx gmbh weinbergweg 23...

SH-SY5Y: transfection of siRNA

VG-01LB-00, VG-01LB-01

Lipocalyx GmbH Weinbergweg 23 06120 Halle Germany viromer-transfection.com viromer-transfection.com

Fig. 1: Knock-down of gene A with increasing RNAi concentrations (6, 12, 25 and 50nM) and optimized Viromer® GREEN quantity

• qPCR analysis of transcript level (Gene A). Normalized to the geometic mean of 4 reference genes.• LUC: RNAi targeting luciferace (Control)• Cells: Viromer treated cells without RNAi sequences• Read-out: 48h post-transfection• No sign of toxicity

Data from Y. Lanny & S. Van der Laan - CNRS, IGH Montpellier (France)

https://viromer-transfection.com/product/sirna-transfection-viromer-green

http://viromer-transfection.com/

Fig. 2: (Fig. 8D and 8E from Prescott et al (2014) J. Cell Sci.) Reduction of SMN protein complex expression in human neuroblastoma SH-SY5Y cells after siRNA transfection with Viromer® GREEN.

„The efficiency of silencingwas extremely high (>90%

of cells)“

SH-SY5Y: transfection of siRNA

VG-01LB-00, VG-01LB-01


Fig. 8. Overexpression of GFP–SMN results in enlarged vesicles with low mobility, whereas reduction of endogenous SmBresults in loss of SMN-rich cytoplasmic structures.(A) Undifferentiated cell from line SHY5mCherrySmB transiently expressing GFP–SMN. mCherry–SmB (i) and magenta in overlay is found in large cytoplasmic structures (arrows) colocalizing with GFP–SMN (ii and green on overlay). Single z-sections from a representative time-lapse sequence. (B) Graphical representation of movements of mCherrySmB vesicles in cells expressing GFP–SMN and untransfected control cells. The velocity of the vesicles between time points is plotted against time, each coloured line represents the track taken by a single vesicle. Total duration of the GFP–SMN-expressing time course is longer owing to the time required for dual-color imaging at each time point. (C) Both mean and maximum track velocities are significantly reduced by overexpression of GFP–SMN. Mean and maximum track velocities, plotted as mean ± s.e.m., are significantly decreased by expression of GFP–SMN (unpaired Student's t-test, n = 89). (D) SmB vesicles persist in cells with reduced levels of SMN or PRMT5. Cells from line SHY5mCherrySmB transfected with siRNA sequences to reduce SMN (siSMN) or PRMT5 (siPRMT5) or non-targeting duplexes (−ve control) contain detectable SmB vesicles (arrows). The efficiency of transfection was extremely high (>90% of cells) as evidenced by the use of siGLO-labelled duplexes targeting cyclophilin D (siGLO, arrows). (E) Depletion of SmBresults in loss of SMN-rich cytoplasmic structures. Cells from line SHY10GFPSMN transfected with siRNA sequences to reduce SmBexpression (siSmB) contain large numbers of nuclear accumulations of GFP–SMN (arrows) but no cytoplasmic accumulations. Cells transfected with siRNA sequences targeting luciferase (−ve control) show the expected pattern of a small number of nuclear accumulations (arrows) and some cytoplasmic accumulations (arrowhead). Cells transfected with sequences to reduce SMN (siSMN) show no nuclear or cytoplasmic accumulations of SMN. In contrast to the result with siSmB, cells from the same line treated with leptomycin B (16 hours LMB) show a large number of cytoplasmic accumulations of GFP–SMN (arrowheads) when compared with control, untreated cells (no LMB), which contain a small number of nuclear accumulations (arrows) in addition to cytoplasmic signal. Scale bars: 10 µm.

http://www.ncbi.nlm.nih.gov/pubmed/?term=Time-resolved+quantitative+proteomics+implicates+the+core+snRNP+protein+SmB+together+with+SMN+in+neural+trafficking

https://viromer-transfection.com/product/sirna-transfection-viromer-green


Cells seeded at 500,000 cells/well (6-well plate format) Standard protocol Incubation time: 48h (observation at 24h: same efficiency for

pEGFP-N2, lower for pNFH-GFP) No change of medium after transfection Approx. transfection efficiency: 80% and 40% respectively

(obtention of stable clones)

pEGFP-N2 (Clonetech®)

pNFH-GFP Letournel et

al, Neurosciences

(2006)

Data from F. Letournel, Institut de Biologie en Santé, Unité Neurobiologie-Neuropathologie, CHU Angers (France)

Note: Cell nuclei stained with DAPI

VR-01LB-00, VR-01LB-01

SH-SY5Y: transfection of plasmid DNA


Fig. 3: Overexpression of GFP after plasmid transfection in human neuroblastoma SH-SY5Y cells with Viromer® RED – test with 2 different plasmids.

http://www.sciencedirect.com/science/article/pii/S0306452205009784

https://viromer-transfection.com/product/plasmid-transfection-viromer-red


VR-01LB-00, VR-01LB-01



Fig. 4: Estimation of cells overexpressing GFP 24h after plasmid transfection in human neuroblastoma SH-SY5Y cells with Viromer® RED – test with Viromer® Positive Controls and a second GFP plasmid.

Data from Y. Lanny & S. Van der Laan - CNRS, IGH Montpellier (France)

Negative Control

Positive Control 1X Positive Control 2X

GFP Plasmid

Viromer RED Controls

Scale x1.5Scale x0.5 Scale x1

Max 70%

Max 40%



https://viromer-transfection.com/product/plasmid-transfection-control-viromer-red-gfp

Fig. 5: Comparison of GFP overexpression after plasmid transfection in human neuroblastoma SH-SY5Y cells with Viromer® RED, FuGene® HD and TransIT-X2®

VR-01LB-00, VR-01LB-01



Data from H. Cynis, Fraunhofer Institute for Cell Therapy and Immunology, Halle (Germany)



VR-BUNDLE-01

Fig. 6: Comparative GFP overexpression after plasmid or mRNA transfection in SH-SY5Y neuroblastoma cells with Viromer® RED Start Positive® Controls



Viromer particles already complexed to pCMV-GFP plasmid (Plasmid Factory) or GFP-mRNA (Trilink)

SH-SY5Y: transfection of plasmid DNA and mRNA

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SH-SY5Y: supplemental infotransfection of standard plasmids vs. minicircles


Minicircles (MC) plasmids from Plasmid Factory and standard pCMV vectors encoding GFP used for complexation with Viromer® RED, Viromer® YELLOW, Lipofectamine® 3000 and Mirus TransIT®-X2

Cells cultured in 96-well plates (10% CO2, DMEM 10% FBS, 50.000 cells/well) 90% confluency at the day of transfection 53 ng and 82 ng of MC are equimolar to 82 ng and 126 ng of pCMV, respectively FACS analysis 24h post-transfection

>> Viromer® RED outcompetes all other transfectants and shows a clear preference for minicircle pDNA

Transfection efficiency(% GFP+ cells)

GFP expression level (mean fluo)

Corresponding microscopy pictures on next page


http://www.plasmidfactory.com/



pCMV 126 ng MC 82 ng

Viromer® RED

Viromer® YELLOW

Lipofectamine® 3000

TransIT®-X2






Same experimental design used with Minicircles (MC) plasmids and standard pCMV vectors encoding Luciferase

>> Viromer® RED and Lipofectamine® 3000 give the highest efficiency with the standard pCMV>> Use of minicircles is advantageous for Viromer® RED and TransIT-X2>> Combination of Viromer® RED and minicircles outperforms all other complexation options






sh-sy5y: transfection of sirna · sh-sy5y: transfection of sirna ... lipocalyx gmbh weinbergweg 23...

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