www.polyplus-transfection.com

News 04 - Spring / Summer 2007

siRNA delivery: a key step for effi cient gene silencing

Come and meet our scientifi c experts:

Visit Polyplus’s booth at the following meetings:

May 30 – June 3, 2007American Society of Gene Therapy 10th Annual Meeting, Seattle WA, USAJuly 8 - 12, 2007Life Sciences 2007Glasgow, UKSeptember 19 - 21, 2007RNAi Europe 2007 Annual Meeting, Barcelona, Spain December 1 – 5, 2007 American Society for Cell Biology 47th Annual Meeting, Washington DC, USA

IntroductionThe RNA interference (RNAi) pathway involved in cellular defense against viral invasion, transposon expansion and post-transcriptional regulation was initially discovered in the late 1990’s by Andrew Fire and Craig Mello1. They showed that double-stranded RNA (dsRNA) induced gene silencing in the nematode Caenorhabditis elegans. This mechanism,

By Anne-Laure Bolcato-Bellemin, Marie-Elise Bonnet, Patrick Neuberg and Patrick Erbacher, Polyplus-transfection SA

Abstract

RNA interference is a powerful mechanism exploited as a tool for gene silencing studies. When introduced by transfection, chemically synthesized siRNA are able to silence exogenous and endogenous genes in mammalian cells both at the mRNA and the protein level. Polyplus-transfection has developed a novel transfection reagent, INTERFERin™, which enables delivery of picomolar levels of active siRNAs into mammalian cells without affecting gene silencing effi ciency. Moreover, we demonstrate that INTERFERin™ can be used on a wide range of mammalian cells including suspension and primary cells without any detectable cellular toxicity.

conserved throughout evolution and observed in most eukaryotes, represents a unique form of post-transcriptional gene silencing. Since these early observations, the natural RNA interference pathway has now become a tool for gene function analysis, gene mapping or signaling pathway studies. In mammalian cells, chemically synthesized siRNAs were shown to specifi cally suppress expression of endogenous and heterologous genes, when transfected with non-viral transfection reagents2.Effi cient siRNA transfection is a key step for accurate functional studies in gene silencing experiments. Furthermore, several independent reports showed that delivery of siRNA is able to activate interferon response and lead to off-target effects3, 4, 5. These studies have emphasized the need for new siRNA delivery reagents allowing effi cient gene silencing at low siRNA concentrations. INTERFERin™ transfection reagent meets these criteria (Figure 1). Here we show that transfection of picomolar concentrations of siRNA with INTERFERin™ leads to highly effi cient gene silencing. An additional characteristic of INTERFERin™ is its low toxicity as it does not affect cellular viability and cell growth. Finally, we describe the simple and robust protocol of INTERFERin™.

Fig. 1. Endogenous Lamin A/C silencing using INTERFERin™. CaSki cells were transfected with 1 nM of 21-mer siRNA duplexes matching the lamin A/C sequence using INTERFERin™. After 48 h, lamin A/C silencing effi ciency was determined by immunofl uorescence microscopy.

In this issue

� > � Article • siRNA delivery: a key step for

effi cient gene silencing

� > � Technical Note • Practical tips to improve your DNA

transfection using jetPEI™

• Tips for successful in vitro RNAi experiments

� > � Article• Advances in protein, antibody and

peptide delivery to live cells

� List of distributors

In vivo literature update• An effi cient method for siRNA

delivery in the brain

www.polyplus-transfection.com2

INTERFERin™ protocolPolyplus-transfection has developed INTERFERin™, a ready-to-use reagent especially formulated for the delivery of chemically synthesized siRNA. The transfection protocol is fast and simple (Figure 2). One day prior to transfection, adherent cells are plated in order to reach 30 to 50% confl uency on the day of transfection. INTERFERin™ is added to siRNA diluted in serum-free medium. After incubation, the siRNA/INTERFERin™ complexes are added to the cells kept in growth medium containing serum and antibiotics. Gene expression analysis is usually performed between 24 to 96 h post-transfection. Specifi c protocols for suspension cells such as THP-1 and K562 are also available.INTERFERin™ is well suited for reverse transfection where the siRNA/INTERFERin™ complexes are prepared directly in the wells and the cells are added subsequently.

Cells such as CHO, HEK-293, RAW 264.7, HT-29, HeLa and A549 Luc were successfully transfected using the reverse protocol.

Potency of INTERFERin™ for gene silencingFollowing the development of a new lipid formulation, transfection conditions were optimized in order to reach the highest gene silencing using the lowest possible siRNA concentration. Delivery of luciferase siRNA (GL3) was performed with INTERFERin™ in A549 Luc cells stably expressing the luciferase gene. A mismatch siRNA (GL2) was used as a control and the luciferase gene expression was assessed 48 hours after transfection. As shown in Figure 3, over 90% inhibition of gene expression was obtained with 1 nM siRNA. This inhibition is specifi c as the control

> Article

siRNA did not induce any silencing. Even at 10 picomolar siRNA, 30% inhibition of luciferase gene expression was still observed.

Comparison of INTERFERin™ with competitorsWe compared, at low siRNA concentrations, the gene silencing effi ciency achieved with INTERFERin™ to that obtained with other commercially available transfection reagents dedicated to siRNA delivery. Figure 4 shows that below 100 pM siRNA, transfection with INTERFERin™ results in the highest gene knockdown. Moreover, overall cell viability was not affected by INTERFERin™ treatment while toxicity was observed with some other reagents (data not shown).

Fig.2. INTERFERin™ fast and simple standard protocol in 24-well plates (per well).

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Fig. 3. INTERFERin™-mediated siRNA transfection inhibits luciferase expression in A549 Luc cells.Cells were transfected in the presence of serum with decreasing concentrations of luciferase siRNA (GL3Luc) or control siRNA (mismatch GL2Luc) duplexes using INTERFERin™. Luciferase expression was measured 48 h after transfection.

Fig. 4. Comparison of INTERFERin™ effi ciency with other siRNA delivery reagents.A549 Luc cells were transfected with decreasing concentrations of GL3Luc siRNA using the reagent indicated according to the manufacturer’s protocol. Luciferase expression was determined 48 h after transfection. The inhibition of luciferase gene expression (%) was normalized to untransfected cells.

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� Dilute siRNA (1 nM) in 100 µl of culture medium (without serum)

Add 2 µl of INTERFERin™, vortex

� Incubate for10 min at RT

� Remove medium and add 0.5 ml of complete medium

� Add siRNA/INTERFERin™ complexes to the cells and incubate at 37°C

� Measure gene expression (between 24 to 96 h).

INTERFERin™

Reagent HReagent RReagent OReagent S

siRNA delivery: a key step for effi cient gene silencing.

www.polyplus-transfection.com3

Effi cient siRNA delivery siRNA delivery is a key step for gene knockdown study. Clearly, poor transfections that introduce low, moderate or high level of siRNA into only a fraction of the cell population may generate erroneous or partial phenotypes leading to inaccurate gene function analyses.

In order to determine siRNA transfection effi ciency, A549 Luc cells were transfected with decreasing concentrations of fl uorescent-labeled siRNA (50 nM to 10 pM) using INTERFERin™. The transfected cells were observed by fl uorescent microscopy between 4 to 24 hours after transfection. Figure 5A clearly shows that 100% of the cells were transfected with the siRNA at concentrations ranging from 50 nM to 10 nM siRNA.

Lower siRNA concentrations such as 1 nM are below detection levels, preventing accurate visualization of fl uorescent siRNA within the cells. Even though fl uorescent-labeling was not detected within the cells, over 90% gene inhibition was still observed at 1 nM siRNA (Figure 5B).

Thus, our data show that the detection of fl uorescent siRNA inside the cells does not correlate with effi cient gene silencing. They also suggest that low amounts of siRNA, even if undetectable, are suffi cient for effective RNA interference. Therefore, we recommend performing optimization studies with functional siRNA rather than with labeled siRNA and to determining effective gene silencing at the mRNA and/or protein levels.

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Fig. 5. Delivery of Rhodamine-siRNA into A549 Luc cells.A549 Luc cells were transfected with various concentrations (50 nM to 10 pM) of Rhodamine-GL3Luc-siRNA with INTERFERin™. The cellular localization of the siRNA was observed by fl uorescence microscopy 24 h after transfection (A). Luciferase expression was measured after 48 h (B).

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Excellent cell viability with INTERFERin™Transfections with amphiphile-based formula-tions tend to have adverse effects on cells.

Thus, in order to achieve high silencing effi ciency without affecting cell viability, it is critical to develop an effi cient reagent and to optimize transfection conditions, in particular the volume of reagent used.

Several cell lines (A549 Luc, HeLa, MCF-7, NIH-3T3, CaSki, SiHa, HEK-293, CHO, and HepG2) were transfected in 24-well plates with 1 nM siRNA and increasing volumes of INTERFERin™ (0.25 to 2 µl per well). Figure 6A shows that upon reduction of the volume of INTERFERin™ from 2 µl to 0.25 µl, the silencing effi ciency is only slightly decreased (from 92% with 2 µl to 70% with 0.25 µl). Thus, the optimal volume of INTERFERin™ in 24-well plates is between 0.75 µl and 2 µl. Even with 2 µl of INTERFERin™ per well, no cellular toxicity was observed in any studied cell lines (Figure 6B). This demonstrates that INTERFERin™ does not affect cell viability when used in optimal conditions.

Effi cient silencing at the mRNA and protein levelsWe next analyzed gene silencing upon siRNA transfection with INTERFERin™, both at the mRNA and protein levels. NIH-3T3 cells were transfected with various vimentin siRNA concentrations. mRNA levels were measured by bDNA (QuantiGene™ Reagent Systems, Panomics) and protein levels were determined by Western blot 48 hours after transfection. Vimentin mRNA levels were drastically reduced (over 80% at 1 nM) after siRNA transfection with INTERFERin™ (Figure 7A). Similarly, we observed a signifi cant decrease in Vimentin protein levels (Figure 7B). Our data demonstrate that siRNA delivery with INTERFERin™ induced silencing both at the mRNA and at the protein levels, as expected in RNA interference.

> Article

siRNA delivery: a key step for effi cient gene silencing.

Fig. 6. Silencing and cell viability with various volumes of INTERFERin™.A549 Luc cells were transfected with 1 nM siRNA GL3Luc and increasing volumes of INTERFERin™ in 24-well plates. Luciferase expression was measured 48 h after transfection (A). Cell lines were transfected with 2 µl INTERFERin™ and 1 nM siRNA (GL3Luc) in 24-well plates. The cell shape was observed by contrast phase microscopy 24 h after transfection as an indication of cellular viability (B).

A549 Luc

CHO

HEK-293HeLa

HepG2MCF-7

NIH-3T3CaSkiSiHa

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Fig. 7. Decrease in mRNA and protein levels with INTERFERin™ in NIH-3T3 cells.NIH-3T3 cells were transfected with vimentin siRNA using INTERFERin™. Non-specifi c siRNA was used as negative control. The vimentin mRNA levels were determined 48 h after transfection by bDNA (A) (QuantiGene™ Reagent Systems, Panomics). The protein levels were measured by Western blot 48 h after transfection (B).

www.polyplus-transfection.com5

Silencing effi ciency in various cell linesTransfection effi ciency and gene silencing may vary depending on both the cell line and the target gene. Therefore, we have adapted transfection conditions for diffi cult-to-transfect cells, when needed.Table 1 presents silencing data obtained with several specifi c siRNAs for a range of cell lines using INTERFERin™. We measured over 90% inhibition of gene expression at the mRNA level with 1 nM siRNA in most adherent cells such as epithelial and fi broblast-like cells. Similar results were obtained in primary cells (1 nM siRNA) and suspension cells (5 nM siRNA).

ConclusionTo date, effi cient siRNA delivery remains a limiting step for gene silencing studies by RNAi. Successful gene silencing depends on several factors including the effective amount of siRNA delivered, the potency of the delivery reagent, the adequate sequence and specifi city of the siRNA as well as the cell viability after transfection. The novel lipid-based transfection reagent INTERFERin™ allows effi cient delivery of siRNA into cells and selective gene silencing at 1 nM siRNA for various target genes in a wide range of cells (e.g. adherent, primary and suspension cells). In addition, no toxicity was observed upon transfection with INTERFERin™, a critical aspect when working with delicate cells. Furthermore, the transfection protocol is extremely simple and does not require medium changes since INTERFERin™ is compatible with serum and antibiotics. Taken together, our data show the great potential of INTERFERin™ to deliver picomolar levels of active siRNA into mammalian cells.

References

1. Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E. and Mello, C. C. (1998) Potent and specifi c genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 806-11.

2. Elbashir A. S., Harborth J., Lendeckel W., Yalcin A., Weber K. and Tuschl T. (2001) Duplexes of 21-nucleotide rNAs mediate RNA interference in cultured mammalian cells. Nature 411: 494-8.

3. Semizarov, D., Frost, L., Sarthy, A., Kroeger, P., Halbert, D. N. and Fesik S. W. (2003) Specifi city of short interfering RNA determined through gene expression signatures. PNAS 100: 6347-52.

4. Sledz, C., Holko, M., de Veer, M., Silverman, R. and Williams, B. (2003) Activation of the interferon system by short-interfering RNAs. Nature Cell Biol 5: 834-9.

5. Fedorov, Y., Anderson, E. M., Birmingham, A., Reynolds, A., Karpilow, J., Leake, D., Marshall, W. S. and Khvorova, A. (2006) Off-target effects by siRNA can induce toxic phenotype. RNA 12: 1188-96.

ADHERENT CELL LINES (1 nM siRNA)A549 Luc Luciferase

> 90%

HeLa GAPDH / Lamin A/CCaSki GAPDH / Lamin A/CMCF7 GAPDH / Lamin A/CNIH-3T3 VimentinRAW 264.7 Eg5SiHa GAPDH / Lamin A/CHepG2 GAPDH 60-70%PRIMARY CELLS (1 nM siRNA)MEF Murine embryonic fi broblasts

GAPDH

> 90%Human fi broblasts GAPDH / Lamin A/C

Human hepatocytes GAPDH

SUSPENSION CELL LINES (5 nM siRNA)K562 GAPDH

> 80%THP-1 GAPDH

Table 1. Successfully transfected cell lines and silencing effi ciencies obtained with INTERFERin™ for several targeted genes.

Reagent Cat. N° SizeNumber of

transfections in 24-well plates

INTERFERin™

409-01 0.1 ml 50-100

409-05 0.5 ml 250-500

409-10 1 ml 500-1000

409-50 5 x 1 ml 2500-5000

Bulk quantities are available upon request.

www.polyplus-transfection.com6

> Technical note

Practical tips to improve your DNA transfection using jetPEI™

Amount of DNA required per well for various cell culture plate formats • 6–well plates: 3 µg DNA and 6 µl jetPEI™

• 24–well plates: 1 µg DNA and 2 µl jetPEI™

• 96-well plates: 0.25 µg DNA and 0.5 µl jetPEI™

Optimize

If your transfection efficiency is low (less than 10 %), try successively the following optimization steps: � Increase the N/P ratio by increasing the volume of jetPEI™

transfection reagent (µl per well). Increase the amount of DNA keeping N/P= 5. � Gently centrifuge the cell culture plate at 280 g for 5 min. If

successful, proceed with optimizing the volume of jetPEI™. � Decrease the volume of medium during transfection by half or

less. Incubate for 4 hours at 37 °C and complete medium to usual volume.

� Combine the centrifugation method, the reduced volume of medium and the optimal volume of jetPEI™.

If you are able to increase the interaction between jetPEI™/DNA complexes and cellular membranes, you are likely to increase complexes uptake without toxicity and thus improve transfection efficiency.

Standard conditions in 24-well plates

1 µg DNA and 2 µl jetPEI™ (equivalent to N/P=5)

The N/P ratio is a measure of the ionic balance of the DNA/ jetPEI™ complexes, which refers to the number of nitrogen residues of jetPEI™ per DNA phosphate.

jetPEI™ standard transfection protocol in 24-well-plates

Example: Optimization of transfection conditions for Monkey CV1 cell line in 96-well plates using the batch protocol.

The batch protocol is a fast and easy procedure: the cells are split and transfected on the same day, saving time and effort.

In this experiment we have tested several amounts of DNA ranging from 30 to 150 ng and N/P ratios of 3 and 5. We also compared two transfection volumes: 200 and 120 µl per well. Please note that for CV1 cells, the transfection effi ciency is considered as signifi cant above 106 RLU/mg of protein.

Results

The standard conditions (in blue) represent transfections in a fi nal volume of 200 µl per well. Reducing the volume during transfection to 120 µl (shown in orange) increases the transfection effi ciency approximately 10 fold for all conditions tested. Still, a plateau in transfection effi ciency is reached with 100 ng of DNA at N/P=5.

For small transfection volumes (in orange), signifi cant transfection effi ciency (above 106 RLU/mg of protein) is obtained with only 60 ng of DNA at N/P=5, while in standard conditions (in blue), such levels of luciferase are achieved with 100 ng of DNA at N/P=3. These data demonstrate the importance of optimizing the transfection volume.

Conclusion

The highest transfection effi ciency of CV1 cells is obtained in the reduced volume of transfection (120 µl) with 100 ng of DNA at N/P=5. This data show that simple optimization steps signifi cantly improve your transfection effi ciency with jetPEI™.

Polyplus-transfection owns the exclusive worldwide

license (#133139, Registre National des

Brevets, 14/05/2003) for transfection of

nucleic acids using polyethylenimine and

derivatives (Boussif et al., 1995).

Reagent Cat. N° Size NaCl solution*

jetPEI™

101-01N 0.1 ml 5 ml101-05 0.5 ml —101-05N 0.5 ml 50 ml101-10 1 ml —101-10N 1 ml 50 ml101-40 4 x 1 ml —101-40N 4 x 1 ml 4 x 50 ml

One ml of jetPEI™ is suffi cient to perform 500 transfections in 24-well plates (1 µg of DNA per well).

* NaCl complex-formation solution included (adapted to proper complex-formation, as indicated in the protocol).

Incubate 24 to 48 h and measure protein expression

q

¢¢

jetPEI™ in 150 mM NaCl

Vortex

DNA in 150 mM NaCl

Incubate 15 min at RT and add to the cells

Transfection of CV1 in 96-well plate using jetPEI™

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60 ng30 ng 100 ng 150 ng

Add

www.polyplus-transfection.com7

> Technical note

Add INTERFERin™ (1-2 µl)

siRNA (1nM) in 100 µl serum-freemedium

Vortex 10 s

Incubate 10 min at RT

Add to cells in 0.5 ml complete medium

Incubate at 37°C

INTERFERin™ is an easy-to-use reagent that allows effi cient delivery of siRNA to cells and robust gene silencing using nanomolar concentrations of siRNA. The protocol for INTERFERin™ could hardly be much simpler; it is fast and easy, whether you use a forward or a reverse protocol. In addition, you can keep your cells in serum and antibiotics during transfection.

Prepare your cells• Use healthy cells, preferably at low

passage.• Plate cells at 30 to 50% confl uency, one

day before transfection.

Prepare the siRNA• When using low siRNA concentrations,

adapt the concentration of the siRNA stock solution accordingly. Prepare a working aliquot allowing you to pipet accurate volumes.

Transfection protocol in 24-well plates

Transfection tips• Vortex INTERFERin™ before use. Ensure

the reagent was kept refrigerated at 4 °C.• Do not incubate the siRNA/INTERFERin™

complexes longer than 30 min.• For small volumes of INTERFERin™, dilute

the reagent 1/5 in water and dispense.• Gently swirl the plate to homogenize after

transfection.

Next day • No need to change medium the day

after transfection for standard cell lines. For sensitive cells, replace the medium 4 to 6 hours after transfection or the next morning.

Tips to increase siRNA silencing• Optimize amount of siRNA used and

volume of INTERFERin™. For primary cells, use higher concentrations of siRNA (i.e. 10, 20 or even 40 nM).

• Use Opti-MEM® to prepare the INTERFERin™/siRNA complexes.

• Reduce the volume of medium on the cells by half.

• Centrifuge the plate at 280 g for 5 min and replace medium after 4 hours.

Tips for successful in vitro RNAi experiments

Your gene of interest• Check the expression profi le of your gene.• Check the turnover of your protein of

interest and the half-life of your mRNA (if known) to determine the best time point for analysis.

• Check various time points after transfection namely at 24, 48, 72 and 96 hours after transfection.

• Determine gene silencing effi ciency at the RNA level and at the protein level.

Check your siRNA• Use high quality desalted siRNA.• Validate your siRNA sequence

(for example in a co-transfection assay).• Use a standard control such as a

housekeeping gene or GAPDH siRNA.• Use a commercially available negative

control such as scrambled siRNA.• Verify siRNA concentration and

annealing.• Ensure the siRNA has been stored

properly.

Tips to reduce toxic effects on primary cells• Replace medium after 4 to 12 hours.• Reduce the volume of INTERFERin™.• Check that the target gene is not involved

in cell viability.

For more information, please conctact our technical support: support @polyplus-transfection.com

Reagent Cat. N° SizeNumber of

transfections in 24-well plates

INTERFERin™

409-01 0.1 ml 50-100

409-05 0.5 ml 250-500

409-10 1 ml 500-1000

409-50 5 x 1 ml 2500-5000

Bulk quantities are available upon request.

www.polyplus-transfection.com8

> Article

Advances in protein, antibody and peptide delivery to live cellsBy Claire Weill, Amélie Weiss, Stéphanie Biri, Jeanne-Françoise Williamson, Adib Abdennaji and Patrick Erbacher, Polyplus-transfection SA

Abstract

The delivery of proteins to live cells offers a powerful alternative as a research tool where other approaches have failed. In this work, we show the effi ciency of PULSin™, a reagent for protein and antibody delivery into cells. PULSin™ promotes effi cient crossing of proteins through the plasma membrane and their release from the endosomes. The delivered protein, in its native form, is then able to diffuse within the cytoplasm and to resume cellular functions. In addition to delivering proteins and antibodies, we now demonstrate that PULSin™ is able to deliver peptides in a wide variety of cells.

Introduction

The intracellular delivery of proteins, antibodies and peptides to the cytoplasm and to the nucleus is usually impeded by the cell membrane. In order to allow facilitated, non-invasive delivery of such biomolecules, we have developed a potent carrier namely PULSin™. This novel reagent acts as follows: the fi rst step of intracellular delivery of biomolecules with PULSin™ is the formation of non-covalent complexes between the biomolecules of interest and the delivery agent. Complexes are subsequently internalized via anionic cell-adhesion receptors1 and released into the cytoplasm where they disassemble. The native protein can thus diffuse throughout the cytoplasm and reach its target organelle to perform its biological function. The process is non-toxic and delivers functional proteins. In addition to delivering proteins and antibodies, PULSin™ is also shown to deliver peptides into a wide variety of cells. Here, we describe a range of applications for intracellular delivery of protein, antibody and peptide. We also discuss protocol optimizations and show the effi ciency of delivery in various cell types.

Applications

Protein delivery

Intracellular protein delivery represents a more direct experimental approach compared to the transfection of DNA and gene expression. In the latter case, protein expression is dependent on effi cient transport of the plasmid to the nucleus and transcriptional regulation. In contrast, biomolecule delivery using PULSin™ allows immediate study of protein function. Furthermore, with PULSin™ technology there is no need to create stable cell lines to study proteins. When required, just deliver your protein of interest into the cells and perform a functional analysis.

A large number of proteins have successfully been delivered intracellularly using PULSin™: namely R-phycoerythrin (RPE), Histone H1, Green Fluorescent Protein (GFP), Bovine Serum Albumin (BSA) and ß-galactosidase. As shown in Figure 1, fl uorescently labeled Histone H1 (AlexaFluor®488, Molecular Probes, France) was mainly detected in the nucleus of HeLa cells using PULSin™. Therefore, upon introduction into the cytoplasm with PULSin™, this nuclear protein is effectively able to cross the nuclear pore and reach the nucleus.

Antibody delivery

Delivery of antibodies to live cells presents a wide range of interesting applications as antibodies are able to recognize proteins within cells and/or to block protein function.

For example, Cassinelli and their collaborators2 showed that anti-phosphorylated Met antibody delivered by PULSin™ into cells induced a signifi cant inhibition of cell migration and invasiveness. The anti-phosphorylated Met antibody

Fig. 1. Delivery of Histone H1-AF®488 into HeLa adherent cells. Histone H1-AF®488 (Molecular Probes, 4 µg) was added to PULSin™ (3 µl) to form complexes. Histone H1/PULSin™ mix was added onto HeLa cells and picture was taken 20 h later.

www.polyplus-transfection.com9

recognizes the activated form of Met in the human lung cancer cell line H460 and was able to reach its target antigenic protein, resulting in the inhibition of Met-dependent downstream signaling.

Peptide delivery

Peptides are biomolecules acting with high specifi city at low concentrations and bioactive peptides such as substrates, inhibitors or modulators can be identifi ed and isolated by library screening or computer modeling. The intracellular role of such peptides is mediated through interactions with intracellular protein targets. Thus, the delivery of peptides into cells not only allows protein function studies but also presents a powerful tool for the development of therapeutic approaches. Such bioactive peptides vary in size, net charge and ratio

of hydrophobic/hydrophilic amino acids. For this work, we have designed three peptide sequences in order to analyze the infl uence (i) of the net charge, (ii) of the size and (iii) of the ratio of hydrophobic and hydrophilic amino acid on delivery effi ciency.

P1 and P2 peptides (Figure 2) are 16 amino acids long and of opposite charges. They include 6 hydrophilic and 5 hydrophobic residues. At physiological pH, P1 is negatively charged while P2 is positive. P3 is a shorter highly hydrophobic peptide of 9 amino acids.

The peptides were fl uorescently labeled with lissamine-rhodamine at the N-terminus and analyzed for their ability to form complexes with PULSin™ under native agarose gel electrophoresis (Figure 3).

As described previously, PULSin™ is a liposomal formulation containing a cationic amphiphile molecule. The anionic peptide P1 and the hydrophobic P3 are both able to form complexes with PULSin™ (Figure 3). However, despite the presence of hydrophobic residues, PULSin™ was unable to form complexes with the positively-charged P2 peptide.

- - - - -

+ + + + +

-

As expected, only P1 and P3 were delivered into HeLa cells using PULSin™ (Figure 4). Effective intracellular delivery was confi rmed in various cell lines (data not shown).

Fig. 2. Schematic representation of peptides. Hydrophobic amino acids are shown in red and hydrophilic residues in blue. Positively or negatively charged residues are also indicated (+ or -, respectively).

Fig. 4. Infl uence of the net charge of the peptide on delivery effi ciency. Peptides P1, P2 or P3 (2 µg) were incubated for 15 min with PULSin™ (4 µl) to form complexes. Peptide/PULSin™ solution was added onto HeLa cells (P1 and P2) or NIH-3T3 cells (P3) and pictures were taken 7 h later using fl uorescent microscopy.

Fig. 3. Analysis of the formation of complexes between peptides and PULSin™. 0.5 µg of fl uorescently labeled peptide was incubated in Hepes buffer (20 mM, pH 7.4) for 15 min at room temperature in the presence (+) or absence (-) of 4 µl of PULSin™, loaded on an agarose gel (2 % in Tris-Acetate 20 mM pH 7.4) and submitted to electrophoresis during 1 h at 70 V/cm. The picture of the agarose gel was taken on a UV-bench. P1, P2 and P3 are the peptides described in Figure 2.

P1

P2

P3

P1 P2 P3— + — + — +

PULSin™

P1

P2

P3

Special limited offer*

discount on PULSin™ !

(0.4 ml size, cat. # 501-04)

*Reference code SPO704 when placing an order.

Offer lasts until July 31, 2007

2020%

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> Article

Advances in protein, antibody and peptide delivery to live cells

Our results show that, neither the size, nor the ratio of hydrophobic/hydrophilic residues within a peptide impair the effi ciency of delivery using PULSin™. However, small highly positively charged peptides can prevent the formation of complexes with PULSin™ and thus fail to be delivered to cells.

Protocol for the delivery of biomolecules such as proteins, antibodies or peptides

From an experimental point of view, since PULSin™ is ready-to-use, the protocol is fast and simple. The experiment is performed as follows: cells are plated in order to reach approximately 70-80% confl uency the day of protein delivery experiment. The protocol is given per well for a 24-well plate: dilute 1 µg of purifi ed protein in 100 µl of Hepes buffer (20 mM, pH 7.4) supplied with PULSin™ in a 1.5 ml eppendorf tube under sterile conditions. Add 4 µl of PULSin™ to the protein. After a brief vortexing step and a short centrifugation, incubate the protein/PULSin™ mix for 15 minutes at room temperature. Wash the cell with 1 ml of PBS and add 900 µl of culture medium without serum.

After addition of 100 µl protein/PULSin™ mix onto each well, the plate is gently homogenized and further incubated at 37°C. After 4 hours, the medium is replaced with fresh medium containing serum. Protein delivery or activity can be analyzed immediately or at later time points.

Another great advantage of PULSin™ is that it is a versatile reagent able to form complexes with various proteins and deliver them to a wide variety of cells, including adherent cell lines, primary cells as well as suspension cells. The delivery effi ciency varies depending on the cell type, but in most cells, high levels of protein delivery can be obtained (e.g. up to 95% for NIH-3T3 cells) as shown in Table 1.

Conclusion

Our data taken together with those of other research groups2,3 demonstrate the effective delivery of low amounts (1 µg or less) of active proteins, antibodies or peptides to live eukaryotic cells without any detectable side effects. Therefore, the protein carrier PULSin™ allows the development of novel applications for cell biologists, in particular for the study of lethal proteins to live cells allowing a tight control of the time course and the amount of protein delivered.

Reagent Cat. N° Size Reactionsin 6-well

Reactionsin 24-well

PULSin™

501-01* 0.1 ml 6 24

501-04* 0.4 ml 24 96

501-16** 4 x 0.4 ml 96 384

* This kit contains 20 µg of R-Phycoerythrin (positive control) and 20 ml of Hepes dilution buffer.** This kit contains 20 µg of R-Phycoerythrin (positive control) and 4 x 20 ml of Hepes dilution buffer.

References

1. Kopatz, I., Remy, J.S. and Behr, J.P. (2004) A model for non-viral gene delivery: through syndecan adhesion molecules and powered by actin. J Gene Med 6(7): 769-76.

2. Cassinelli, G., Lanzi, C., Petrangolini, G., Tortoreto, M., Pratesi, G., Cuccuru, G., Laccabue, D., Supino, R., Belluco, S., Favini, E., Poletti, A. and Zunino, F. (2006) Inhibition of c-Met and prevention of spontaneous metastatic spreading by the 2-indolinone RPI-1. Mol Cancer Ther 5(9): 2388-97.

3. Leshchyns’ka, I., Sytnyk, V., Richter, M., Andreyeva, A., Puchkov, D. and Schachner, M. (2006) The adhesion molecule CHL1 regulates uncoating of clathrin-coated synaptic vesicles. Neuron 52(6): 1011-25.

Table 1. Effi ciency of R-phycoerythrin delivery using PULSin™ in selected cells.

Adherent cell lines3T3 L1 60-80% HepG2 20%A549 80% MCF-7 50-60%BHK-21 30-40% MEF 80%CaSki 80-90% MLE-15 60-75%CHO 80-90% NIH-3T3 90-98%CV-1 50% RAW 264.7 40-50%HEK-293 45-55% S2 30-40%HeLa 80-90% SiHa 60-70%HepaRG 60-70%

Suspension cell linesHEK-293 30-40% K 562 20-30%Jurkat 70% THP-1 10%

Adherent primary cellsHuman fi broblasts 60-70%Human hepatocytes 50-40%Human keratinocytes 55-70%Human visceral preadipocytes 60-75%Human umbilical vein endothelial cells (HUVEC)

80%

Special limited offer*

discount on PULSin™ !

(0.4 ml size, cat. # 501-04)

*Reference code SPO704 when placing an order.

Offer lasts until July 31, 2007

2020%

www.polyplus-transfection.com11

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News 04 - Spring / Summer 2007

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Editors : A.L. Monjanel and J.F. WilliamsonGraphics and Layout : S. Nikolic

In vivo literature update

An effi cient method for siRNA delivery in the brainSeveral papers have reported successful delivery of siRNA in the brain using a combination of jetSI™ 10 mM and DOPE (20mM) in a 1:2 molar ratio. Upon intracerebroventricular and hypothalamic injections, jetSI™ 10 mM /DOPE/siRNA complexes allowed signifi cant knockdown of various genes of interest.

Froidevaux et al. (2006) EMBO Rep 7: 1035-9. Guissouma et al. (2006) Neurosci Lett 406: 240-3.Kumar et al. (2006) PLOS Med 3: 0505-1.

TRADEMARKSPolyplus-transfection, jetPEI, jetSI, Fecturin, PULSin and INTERFERin are registered trademarks of Polyplus-transfection SA.

LICENCING INFORMATIONPolyplus-transfection owns the exclusive worldwide license (#133139, Registre National des Brevets, 14/05/2003) for the transfection of nucleic acids using polyethylenimine and derivatives (Boussif et al., 1995).

Let us know about your Let us know about your Publications !Publications !

If you have published a paper with Polyplus reagents, we would love to hear from you. Please send us a copy of your work to support@polyplus-transfection.com and we will send you a complimentary USB 2.0 Hub !