2009/08/07

INTERFERin®

Classé dans : siRNA Transfection — Mots-clefs : — admin @ 11:52
Ordering information
Catalog Number Amount of reagent
409-01 0.1 ml
409-10 1 ml
409-50 5 x 1 ml
1 ml of INTERFERin® is sufficient to perform 500-1000 transfections in 24-well plates.Bulk sizes available, please contact us.

 
More valuable data using less siRNA

INTERFERin®-mediated delivery of GL3Luc siRNA to A549Luc cells stably expressing the GL3 luciferase gene shows selective and highly efficient knockdown of gene expression. Amazingly, 50% silencing is still achieved at 10 pM siRNA in the presence of serum (Fig. 1). Using nanomolar siRNA concentrations results in more accurate data as it avoids unwanted off-target effects often associated with reagents requiring higher amounts of siRNA 1, 2.

INTERFERin-fig1 Fig 1. INTERFERin®-mediated siRNA transfection inhibits luciferase expression in A549-GL3Luc cells. Cells were transfected in 24-well plates in the presence of serum with decreasing concentrations of Luciferase siRNA (GL3Luc) duplexes using INTERFERin®. Luciferase expression was measured after 48 h. No inhibition was observed with control siRNA duplexes (mismatch GL2Luc, data not shown).

Transfection of 1 nM siRNA duplexes targeting endogenous lamin A/C with INTERFERin® drastically reduces lamin gene expression to barely detectable level (Fig. 2).

INTERFERin-fig2

Fig 2. 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 efficiency was determined by immunofluorescence microscopy.

 
Over 90% gene silencing

For adherent cell lines or primary cells, 1 nanomolar or less siRNA concentration are sufficient to obtain more than 90 % gene silencing for a variety of genes. For difficult-to-transfect suspension cell lines, 80% silencing can still be reached by INTERFERin® using 5 nM siRNA concentration (Table 1).

Table 1. Successfully transfected cell lines and silencing efficiencies obtained with INTERFERin®.

 
Superior transfection results - Less toxic

INTERFERin® was compared to three other reagents active at nanomolar concentration. INTERFERin® was clearly the most efficient at the lowest concentrations (Fig. 3).

INTERFERin-fig3 Fig 3. Comparison of INTERFERin® efficiency with three other siRNA delivery reagents. A549-GL3Luc cells were transfected in the presence of serum with decreasing concentrations of GL3Luc siRNA duplexes using the reagent indicated according to the manufacturer’s protocol. Luciferase (GL3Luc) expression was measured after 48 h.

In the same experiment as above (48 h after transfection at 1 nM siRNA), cells transfected with INTERFERin® or with reagent H appeared healthy, while toxicity was observed with reagent S (Fig. 4).

INTERFERin-fig4

Fig 4. Comparison of cell morphology 48 h after siRNA delivery using INTERFERin® or competitor reagents. A549-GL3Luc cells were transfected in the presence of serum with 1 nM of GL3Luc siRNA duplexes using INTERFERin® or competitors S or H according to the manufacturer’s protocol.

 
Easy standard protocol

INTERFERin® is ready to use and the protocol is straightforward. 1 nM siRNA concentration is recommended as a starting concentration for silencing of most genes and cell types (Fig. 5).

  • INTERFERin® is compatible with serum and antibiotics.
  • Forget time consuming medium changes and washes. INTERFERin® can be left on the cells without any adverse effects.

Fig 5. INTERFERin™ standard protocol for 24-well plate.

 INTERFERin-fig5

 
References

1. Semizarov D. et al. (2003) Specificity of short interfering RNA determined through gene expression signatures. Proc. Natl Acad. Sci. USA 100: 6347.

2. Persengiev S.P. et al. (2004) Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs. RNA 10:12.

 
VIDEO: siRNA Transfection using INTERFERin®

 

jetPEI®-Macrophage

Classé dans : Cell Specific, Gene expression, Transfection Reagents — Mots-clefs : — admin @ 11:52
Ordering information
Catalog Number Amount of reagent Amount of NaCl solution
103-01N 0.1 ml 5 ml
103-05N 0.5 ml 50 ml
Complexes formation is perfectly performed with NaCl solution.
0.5 ml of jetPEI®-Macrophage is sufficient to perform 250 transfections in 24-well plates.

 
Transfection of primary human macrophages

As shown in Figure 1, jetPEI®-Macrophage allows successful transfection of macrophages derived from monocytes maturated for 7 days in presence of GM-CSF.

jetPEI-Macrophage-fig1 Figure 1. Human macrophages expressing Beta-Galactosidase after transfection using jetPEI®-Macrophage.

 
Higher transfection efficiency compared to the versatile jetPEI™

Primary human macrophages and murine RAW 264.7 cells express significantly higher protein levels when transfected with jetPEI®-Macrophage (Fig. 2).

jetPEI-Macrophage-fig2 Figure 2. Comparative efficiency of jetPEI®-Macrophage versus jetPEI™ on primary human macrophages and murine RAW 264.7 cells in 24-well plates. Primary human macrophages were transfected in the presence of 100 U/ml GM-CSF and 10% serum, using 1 µg pCMVLuc and 2 µl transfection reagent. Murine RAW 264.7 cells were transfected using 2 µg pCMVLuc and 6.4 µl transfection reagent. Luciferase activity was measured 24 h post-transfection.

 
Easy-to-use protocol

The jetPEI®-Macrophage protocol is as simple as that of jetPEI®: Mix the DNA with the reagent to form complexes and simply add the mixture to the cells. jetPEI®-Macrophage is compatible with serum and antibiotics, thus eliminating the need for media changes. Protein expression is determined 24 h to 72 h post-transfection.

 
References

1. Gregoriadis G. (1998) Genetic vaccines: Strategies for optimization. Pharmaceut Res 15, 661-670

2. Condon C., S. C. Watkins, C. M. Celluzzi, K. Thompson and L. D. Falo (1996) DNA-based immunization by in vivo transfection of dendritic cells. Nature Med 2, 1122-1128

3. Erbacher P., M. T. Bousser, J. Raimond, M. Monsigny, P. Midoux and A. C. Roche (1996) Gene transfer by DNA/glycosylated polylysine complexes into human blood monocyte-derived macrophages. Hum Gene Ther 7, 721-729

 

jetPEI®-Hepatocyte

Classé dans : Cell Specific, Gene expression, Transfection Reagents — Mots-clefs : — admin @ 11:50
Ordering information
Catalog Number Amount of reagent Amount of NaCl solution
102-01N 0.1 ml 5 ml
102-05N 0.5 ml 50 ml
Complexes formation is perfectly performed with NaCl solution.
0.5 ml of jetPEI®-Hepatocyte is sufficient to perform 160 transfections in 24-well plates.

 
Higher transfection efficiency compared to the versatile jetPEI™

Compared to our standard jetPEI™ transfection reagent, jetPEI®-Hepatocyte provides significantly higher transfection efficiencies on human hepatocarcinoma cells HepG2 as well as on the difficult-to-transfect primary human hepatocytes (Fig. 1).

jetPEI-Hepatocyte-fig1 Figure 1. Comparative efficiency of jetPEI®-Hepatocyte versus jetPEI™. HepG2 (50,000 cells in 24-w) and primary human hepatocytes (100,000 cells in 24-w) were transfected using 3.2 µl of both reagents with 1 µg of pCMVEGFPLuc, in 1 ml of complete medium containing 10% of serum. The luciferase activity was determined 48h post-transfection. Transfection efficiency was expressed as relative efficiency obtained with jetPEI® compared to jetPEI®-Hepatocyte.

 
Easy-to-use protocol

jetPEI®-Hepatocyte protocol is as simple as that of the jetPEI®: Mix DNA with the reagent to form complexes and simply add the mixture to cells. jetPEI®-Hepatocyte is compatible with serum1 and antibiotics. No media changes or washes are required. Transgenic protein expression is determined 24 h to 72 h post-transfection.

 
50% transfection efficiency and excellent viability in primary hepatocytes

Primary human hepatocytes 24 h and 48 h after transfection using jetPEI®-Hepatocyte of the luciferase plasmid pCMVEGFPLuc show 50% transfection efficiency. In addition, cells look healthy (Fig. 2).

jetPEI-Hepatocyte-fig2 Figure 2. Primary human hepatocytes expressing GFP 24 h and 48 h after transfection using jetPEI®-Hepatocyte. Primary human hepatocytes (100,000 cells in 24-w) were transfected with 1 µg of pCMVEGFPLuc at N/P ratio of 8, in 1 ml of complete medium containing 10% of serum. Cells were visualized by fluorescence microscopy.

 
Transfect easily hepatocyte-derived cell lines

The most popular hepatocyte-derived cell lines BNL-CL.2 and HepG2 were successfully transfected using jetPEI®-Hepatocyte and commonly show approximately 50 % and 30% transfection efficiency, respectively (Fig. 3 and 4).

jetPEI-Hepatocyte-fig3 Figure 3. BNL-CL.2 cells expressing ßgalactosidase after transfection using jetPEI®-Hepatocyte. BNL-CL.2 cells (50,000 cells in 24-w) were transfected using 3.2 µl of reagent with 1 µg of pCMVEGFPLuc, in 1 ml of complete medium containing 10% of serum. ?-galactosidase gene expression was visualized by X-gal staining after 24h.
jetPEI-Hepatocytefig4 Figure 4. HepG2 cells expressing GFP 72 h after transfection using jetPEI®-HepatocyteHepG2 cells (50,000 cells in 24-w) were transfected using 3.2 µl of reagent with 1 µg of pCMVEGFPLuc, in 1 ml of complete medium containing 10% of serum. Cells expressing GFP protein were visualized by fluorescence microscopy.

 
Mechanism of transfection using jetPEI™-Hepatocyte

jetPEI®-Hepatocyte is a galactose-bearing linear polyethylenimine designed to enhancethe transfection of cells expressing galactose-specific membrane lectins, such as hepatocytes that express the asialoglycoprotein receptor (ASGP-R or Gal/GalNAc receptor). Cell targeting is the result of binding of the galactose residues to the specific cell-surface receptors, leading to internalization of the DNA complexes. Galactose-bearing PEI has also been used to target oligonucleotides to hepatocytes in cell culture2.

 
References

1. Zanta M. A., O. Boussif, A. Adib and J. P. Behr (1997) In-Vitro Gene Delivery to Hepatocytes with Galactosylated Polyethylenimine. Bioconj. Chem. 8, 839-844

2. Kren B. T., P. Bandyopadhyay and C. J. Steer (1998) In-Vivo Site-Directed Mutagenesis of the Factor-IX Gene by Chimeric RNA/DNA Oligonucleotides. Nature Med 4, 285-290

 

jetPEI®

Classé dans : DNA HTS — Mots-clefs : — admin @ 11:48
Ordering information
Catalog Number Amount of reagent Amount of NaCl solution
101-01N 0.1 mL 5 mL
101-10N 1 mL 50 mL
101-10 1 mL -
101-40N 4 x 1 mL 4 x 50 mL
101-40 4 x 1 mL -
101B-010N 10 mL 2 x 250 mL
101B-010 10 mL -

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

One ml of jetPEI® is sufficient to perform 2 000 transfections in 96-well plates.

Bulk quantities are available upon request (50 mL, 100 mL and 1L).

Please contact us.

 
3 protocols to suit your application

The reverse protocol is the most appropriate when transfecting a pool of genes, such as a DNA library (Fig. 1). In this protocol, the jetPEI®/DNA complexes are prepared or deposited in the wells prior to addition of the cells. Complexes are stable for up to 4 hours (Fig. 2).

The batch protocol was developed to prepare a homogeneous pool of transfected cells. For this purpose, the cells are transfected just after trypsinization, while still in suspension. This protocol is prefered for drug screening applications and allows rapid processing, one day faster than the forward protocol.

In the forward protocol, the cells are split the day before transfection and the jetPEI®/DNA complexes are added to the adherent cells.

HTS-jetPEI-Fig1

Figure 1. jetPEI® reverse transfection protocol for HTS application

 
Robust transfection complexes

Complexes formed with the water-soluble polymer jetPEI® and DNA allow efficient transfection for up to 4 hours, in contrast to lipid-based reagents and calcium phosphate. Thus they allow plenty of time to dispense the complexes into the plates (Fig. 2).

HTS-jetPEI-Fig2 Figure 2. Effect of complex formation incubation time on transfection efficiency with jetPEI®. HEK 293 cells were transfected in 96-well plates with pCMVLuc and jetPEI™ following the reverse transfection protocol. Luciferase activity was measured after 24 h.

 
Batch to batch reproducibility

HTS DNA transfection using jetPEI® gives highly consistent transfection efficiency from batch-to-batch (Fig. 3).

HTS-jetPEI-Fig3 Figure 3. Batch-to-batch reproducibility using jetPEI®. For each lot, HeLa cells were transfected in triplicate in the presence of serum in triplicate using the standard protocol in 24-well plate.

 
Efficient in a wide range of cell types

jetPEI® successfully delivers genes to various adherent and non-adherent cell lines, as well as primary cells (Table 1). Over 550 publications using jetPEI® can be found in the Product Citation Database on the Polyplus website. In addition, a Cell Transfection Database gives specific transfection conditions for over 400 cell lines and primary cells.

HTS-jetPEI-Table1 Table 1. Some common cell lines and primary cells successfully transfected using jetPEI®.

 
Superior transfection results

jetPEI® was compared to several other popular transfection reagents (Fig. 4). jetPEI® was found to offer the best performance: high efficiency and low variability (small standard deviation).

HTS-jetPEI-fig4 Figure 4. Transfection efficiency of a series of commercial reagents. HeLa cells were transfected in 24-well plates in the presence of 10% serum, using 1 µg pCMV-luciferase according to the manufacturers’ protocols. Luciferase expression was measured 24 h after transfection.

 
Read more about jetPEI®

PDF Technical note: Fast, efficient and reproductible DNA transfection using the jetPEI® HTS protocol.

 

Transfection in Regular Media

Classé dans : Bioproduction, Protein and Virus Production in Mammalian Cells — Mots-clefs : — admin @ 10:58
Ordering information
Catalog Number Amount of reagent Amount of NaCl solution
101-01N 0.1 ml 5 ml
101-10N 1 ml 50 ml
101-10 1 ml -
101-40N 4 x 1 ml 4 x 50 ml
101-40 4 x 1 ml -
101B-010N 10 mL 2 x 250 mL
101B-10 10 mL -
One ml of jetPEI™ is sufficient to perform 500 transfections in 24-well plates.Large pack sizes available upon request (50 mL, 100 mL and 1L). Please contact us.

 
Reliable recombinant protein and virus production

For large scale production of recombinant proteins such as human therapeutic molecules including proteins, monoclonal antibodies, vaccine antigens and viruses  (rAAV, r-Adenoviruses and r-Lentivirus) by transient or stable transfection, jetPEITM is  the reagent of choice (Fig. 1).

Fig 1: One liter and one ml of jetPEI&trade
 Figure 1: One liter and one ml of jetPEI™

By ensuring a robust, high quality DNA transfection, it leads to efficient and sustained protein expression levels and most importantly guarantees batch to batch reproducibility.

 
Qualified for cGMP virus manufacturing and protein production

With a department dedicated to qualified reagents, Polyplus is able to supply any reagent grade you may require. Furthermore, the QC provided can be tailored to suit any protein production laboratory.

 
Large scale protocols and technical support

Polyplus-transfection provides prime and personalized service including scientific support to suit the pharmaceutical industry requirements.

If you wish to obtain the protocols for 1L of culture or more please contact the Polyplus scientific support specialists.

 
Protein and virus production in Flasks

Polyplus-transfection jetPEI™ is also well-suited for routine virus and protein production in Flasks. For example, it is recommended by Corning for transfection of CHO and HeLa cells in HYPERflasks®.

 
Fully-licensed PEI for Transfection

Polyplus-transfection is the worldwide exclusive licensee of PEI (Polyethylenimine) and derivates for stable and transient transfection of cells.
The purchase of jetPEI™ conveys the buyer right to perform transfection using jetPEI in biomanufacturing applications.

« The use of polyethylenimine (PEI) or polypropylenimine (PPI) or cationic polymers similar in structure thereto for transfecting cells, as well as compositions comprising these cationic polymers and at least one nucleic acid, are the subject matter of U.S. Patent No. 6,013,240, EP Patent No. 0770140 and foreign equivalents, for which Polyplus-transfection™ is the worldwide exclusive licensee. »

For more information, see Licenses.

 

2009/08/06

in vivo-jetPEI®

Classé dans : DNA/siRNA Delivery — Mots-clefs : — admin @ 16:16
Ordering information
Catalog Number Amount of reagent Amount of glucose solution
201-10G 0.1 ml 10 ml
201-50G 0.5 ml 2 x 10 ml
0.1ml of in vivo-jetPEI® is sufficient to perform up to 20 intravenous injections in mouse with 50 µg of DNA.Formation of complexes with the provided glucose solution produces nanoparticules adapted to in vivo injections.Bulk quantities and GMP grade are available upon request. Please contact us.

 
Successful delivery of DNA, siRNA and oligonucleotides in vivo

With recent advances in therapeutics exploiting the growing number of potential nucleic acid molecules used as drugs, in vivo-jetPEI® has become the non-viral delivery reagent of choice for efficient and reproducible nucleic acid delivery in animal models and in the clinic.

in vivo-jetPEI® was initially developed to deliver DNA and oligonucleotides in order to mediate gene expression in various tissues upon in vivo administration. For example In vivo imaging shows high level of gene expression in the lung upon intravenous injection of a Luciferase encoding plasmid in mice (Fig.1 and Table 1). Depending on the route of administration, in vivo-jetPEI®-mediated gene expression was also observed in the brain, liver, pancreas, spleen, kidney, heart, bladder, skin, retina, artery, etc.

invivo-jetPEI-fig1 Figure 1. Systemic delivery using in vivo-jetPEI®. Bioluminescent imaging of luciferase expression in living Balb/C mouse using a cooled camera 24 h after gene delivery. pCMVLuc (50 µg) was complexed with in vivo-jetPEI® in 400 µl of 5% glucose solution and injected into the tail vein. Courtesy J.L. Coll.

 
Multiple modes of administration in many species

The stability of in vivo-jetPEI® DNA complexes allows the use of numerous routes of administration shown in Figure 2. The targeted organs however depend mainly on the injection route.

Routes and target organ:

Upon intravenous injection, in vivo-jetPEI®-mediated DNA delivery leads to gene expression in the lung (Fig 1 and Fig 3) liver, pancreas, spleen, kidney, heart, bladder and artery. In addition, in vivo-jetPEI® is also well-adapted for local delivery such as application onto the skin, intratumoral, intracerebral or intra-articular injections (see Table 1). For experimental conditions using in vivo-jetPEI™, see:

PDFTechnical Note: Guidelines to set up your gene delivery experiments in mice

Literature references are available in the Product citations online database.

invivo-jetPEI-fig2 Figure 2. Successful delivery routes in mouse using in vivo-jetPEI®.

Animal models:
in vivo-jetPEI® was successfully used to deliver nucleic acids to a wide range of species including mouse, rat, guinea pig, duck, rabbit, monkey, goat, sheep, chicken, quail, hamster, cow, tadpole, shrimp and fish. As a result, the experimental set up can be adapted to most species. Our technical support team will be pleased to assist you in order to adjust the reagent to nucleic acid ratio according to your model.

PDFTechnical Note: In vivo publications with Polyplus reagents using the most common administration routes

PDFTechnical Note: In vivo publications with Polyplus reagents by target organ/tissue

 
RNAi with in vivo-jetPEI®

Being versatile and robust, in vivo-jetPEI® is a reagent of choice to inhibit gene expression and to perform RNA interference in selected organs using synthetic siRNA including siRNA with chemically modified nucleobases and Sticky siRNA™, a novel design of siRNA leading to higher gene silencing. Being very potent for DNA delivery, in vivo-jetPEI® is suitable for shRNA and miRNA delivery in vivo. Specific inhibition of protein synthesis can be achieved by delivering siRNA, antisenses oligonucleotides or ribozymes with in vivo-jetPEI® in animal models (Fig.3). Recently, in vivo-jetPEI® and derivatives were successfully used as delivery reagents for anti-cancer therapy by RNA interference (Table 1).

invivo-jetPEI-fig4 Figure 3. RNA interference in the lung with in vivo-jetPEI®. pCMVLuc (40µg) was co-transfected with a control siRNA (upper) or with 10 µg of specific anti-Luc siRNA (lower). The complexes were injected into the tail vein of nude mice. Luciferase gene expression was monitored in living mice 24 h later by bioluminescence imaging using a cooled CCD camera.

shRNA
Since in vivo-jetPEI® was developed for DNA delivery, it has been used successfully for shRNA approaches for RNA interference. Interestingly, it was used for gene silencing in the liver (George and Tsutsumi (2007), Gene Ther 14:890; Parajpe et al. (2007),Hepatology 45:147). Another interesting application in keeping with recent RNAi therapeutic developments in the clinic is the use of shRNA for RNAi in the retina. The method has been optimized by Liao and collaborators (Liao et al. (2007), BioTechniques 42: 285). Furthermore, in vivo-jetPEI® is also suitable for shRNA approaches for RNAi in the brain (Hassani et al. (2007), NAR 35:e65).

siRNA
For siRNA delivery in vivo, several groups have also used in vivo-jetPEI® successfully. This reagent is suitable for any siRNAs whether they include chemically modified nucleobases or not. Current successfully targeted organs in mice include vacsular endothelial cells (Choi et al., (2008) J Biol Chem 283; 20186 ), skin (Murase D., et al., (2009) J Biol Chem 284; 4343), subcutaneous xenograft tumors (Storci G., (2008) J Pathol 214; 25; Urban-Klein B. et al., (2005) Gene Ther. 12; 461, lung (Lively et al., (2008) J Allergy Clin immunol, 121;88) and dendritic cells (Poeck H., (2008) , Nature Med 14;1256; Cubillos-Ruiz (2009) J. Clin. Invest. in press).To consult all references see Table 1 or browse the product citation database.

STICKY SIRNA™ (ssiRNA): a novel approach
In order to improve siRNA delivery in vivo, Polyplus transfection has recently developed a novel type of siRNA. By including longer overhangs within the siRNA, we have generated STICKY siRNA™ (ssiRNA) that are able to form concatemers in the presence of in vivo-jetPEI®, thereby mimicking the structure of DNA and thus enhancing siRNA transfer. This design was shown to improve in vitro siRNA delivery and gene silencing efficiency (Bolcato-Bellemin (2007), PNAS 104:16050). More recently we have shown that intraperitoneal injection of STICKY siRNA™ cyclinB1 in mice using in vivo-jetPEI® in a PC-3 tumor model, lead to an inhibition of tumor metastasis compared to mismatch and control. Moreover, mice survival is increased dramatically with STICKY siRNA™ delivered by in vivo-jetPEI®.

plus-bleu For more information see STICKY SIRNA™

 
No detectable inflammatory response

Linear PEI such as in vivo-jetPEI® does not induce any significant pro-inflammatory response after systemic injection of DNA or siRNA, especially when compared to other highly immunogenic reagents such as branched PEI or other lipid transfection reagents (Kawakami et al. (2006), J Pharmacol Exp Ther 317:1382).

Bonnet and collaborators also showed that there was no induction of major pro-inflammatory cytokines such as  TNF-alpha, IL6, IL12/IL23 and IL-1beta upon DNA and siRNA IV delivery using in vivo-jetPEI®. Furthermore no increase in sera levels of hepatic enzyme were detected, suggesting that in vivo-jetPEI does not induce hepatotoxicity (Fig. 4, Bonnet et al. (2008), Pharm Res, 25:2972). In addition, linear PEI such as in vivo-jetPEI® fails to generate neutralizing antibodies, thus permitting repeated administrations (Garzon et al. (2005), Vaccine 23:138).

invivo-jetPEI-fig5-1invivo-jetPEI-fig5-2 Figure 4 : Serum concentration of TNF-α, IL12/IL23, IFNγ and IL6 following intravenous nucleic acid delivery using in vivo-jetPEI™ (N/P=8) respectively 1h, 6h, 12h and 6 h after delivery. The negative control consists of an IV injection of 5% glucose; the positive control an IP injection of 50 μg E.Coli LPS. (A) 40 μg DNA and (B) 40 ug siRNA were delivered with or without in vivo-jetPEI®.

 
Reproducible results

Gene delivery using in vivo-jetPEI® is reliable providing reproducible data (Fig 5.) with limited toxicity in contrast to other non-viral delivery methods. The reproducibility is linked to the unique properties of in vivo-jetPEI®. In 5% glucose, in vivo-jetPEI® condenses nucleic acids into stable nanoparticles of ca. 50 nm in diameter (Fig 6). As a result, aggregation of blood cells is reduced compared to other reagents (Kircheis et al. (2001), Gene Ther 8: 28), thereby preventing a restriction in diffusion within tissues, erythrocyte aggregation and microembolia. These nanoparticles are sufficiently small to diffuse within the tissues and enter the cells by endocytosis. At the cellular level, in vivo-jetPEI® possesses the unique feature of facilitating escape from the endosome using a proton sponge mechanism (Akinc et al. (2005), J Gene Med 7: 657). It also favors crossing of the nuclear membrane (Brunner et al. (2002), Mol Ther 5: 80).

invivo-jetPEI-fig6 Fig. 5. Systemic delivery using in vivo-jetPEI®. pCMVLuc (50 µg) was complexed with in vivo-jetPEI® in 5% glucose and injected retro-orbitally. After 24 h, luciferase gene expression was assessed in the lung (n=8). Viability of mice was 100%.
invivo-jetPEI-fig7 Figure 6. in vivo-jetPEI® forms small spherical particles with plasmid DNA. in vivo-jetPEI®/DNA complexes are prepared in 5% glucose solution at N/P ratio of 10. Complexes were added on a carbon covered grid and stained with uranyl acetate. Observation was carried out under a TEM. Bar is 100 nm. Complexes produced in glucose solution are discrete spheres having a mean size of 50 +/- 30 nm (Courtesy J-S Remy, Laboratoire Chimie Génétique, CNRS UMR 7514, Illkirch, France).

 

2009/08/05

jetPRIME®

Classé dans : Broad Spectrum — Mots-clefs : — admin @ 18:13
Ordering information
Catalog Number Amount of reagent Amount of buffer
114-01 0.1 ml 5 ml
114-07 0.75 ml 60 ml
114-15 1.5 ml 2 x 60 ml
114-75 5 x 1.5 ml 10 x 60 ml
114-75C 5 x 1.5 ml 120 ml 5X conc.
1.5 ml is sufficient to perform ca.375 transfections in 6-well plates.Bulk quantities are available upon request.Please contact us.

 
Superior DNA transfection efficiency

Superior transfection efficiencies ranging between 70 and 90% were obtained when using jetPRIME® reagent versus the top competitor’s reagent for several commonly used cell lines. (Fig. 1).

jetPRIME vs L2K vB Fig. 1. Transfection efficiency assessed by FACS analysis in various cell lines 24 h following transfection in 24-well plates according to the manufacturer’s recommendation for competitor L2K  and 0.5 µg plasmid, 1 µl reagent per well for jetPRIME®.

Many other cell lines of various origins, as well as primary cells, are transfected with unusually high percentages (Table 1).

List Cells jetPRIME2 Table 1. Transfection efficiency of various cell types using jetPRIME®. The percentage of GFP-positive cells was determined by FACS analysis 24 h after transfection.

 
Economical: less DNA and less reagent

jetPRIME® is such a powerful in vitro transfection reagent that only requires a small amount of reagent and plasmid DNA (Table 2), making it very economical.

jetPRIME-table-vol-vs-L2K

Table 2. Amounts of DNA and reagent (jetPRIME® and competitor) added per well in 6-well plate for transfection according to manufacturers’ recommendations.

In addition to reducing costs, using less DNA also minimizes adverse cytotoxic effects triggered by transfection. Hence, jetPRIME® is the reagent of choice for high transfection efficiency with excellent cell viability.

 
Better cell viability

jetPRIME® is extremely gentle on cells during transfection leading to increased cell viability (Fig. 2) and improved transfection results. Cells transfected with jetPRIME® are healthy, while major cytotoxicity is observed with competitor.

jetprimefig31 jetprimefig32 Fig. 2. Phase contrast microscopy of HeLa cells 24 h after transfections performed according to the manufacturer’s recommendations for each reagent.

 
Excellent gene silencing

jetPRIME® leads to over 90% knockdown of endogenous gene expression in a variety of cell lines. For example, jetPRIME®-mediated transfection of HeLa cells with 10 nM siRNA duplexes targeting endogenous lamin A/C in HeLa cells drastically reduces lamin A/C gene expression to barely detectable level (Fig. 3).

jetprimefig41 jetprimefig42 Fig. 3. Endogenous lamin A/C silencing using jetPRIME®. HeLa cells were transfected with 10 nM of 21-mer lamin A/C siRNA.
After 48 h, lamin A/C silencing was assessed by immunofluorescence microscopy using an antibody against lamin A/C.

 
Cotransfection of DNA & siRNA

jetPRIME® is well suited for DNA and siRNA cotransfection experiments. It shows highly efficient gene silencing in a variety of cell lines with very low toxicity. Over 90% silencing is achieved in adherent cells, using 10 nM siRNA (Fig. 4).

jetprimefig5

 Fig. 5. Exogenous luciferase gene silencing in several cell lines after DNA & siRNA cotransfection using jetPRIME® performed with 400 ng p4CMV-Luc and 10 nM of luciferase siRNA per well in 6-well plates.

 
shRNA and miRNA plasmid transfection

In mammalian cells, RNA interference can be achieved by other means than synthetic siRNA. Another approach consists in using plasmid-based methods. It has been reported that efficient gene silencing can be obtained with small hairpin RNA (shRNA) plasmids. For transfection of such plasmids, jetPRIME®, our versatile DNA transfection reagent, is recommended.

When studying miRNA (micro-RNA) using a plasmid-based approach, the standard transfection method using jetPRIME® is applicable and ensures reliable transfection efficiency.

 
Convenient protocol

jetPRIME® is an easy-to-use transfection reagent (Fig. 5):

• Fast and easy to scale up and down

• Compatible with serum and antibiotics


Fig. 5. jetPRIME™ convenient protocol for DNA, siRNA and co-transfection of DNA and siRNA.
jetprime-fig6

 
VIDEO: DNA Transfection using jetPRIME®

 

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