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0.1ml of ligand-conjugated in vivo-jetPEI™ is sufficient to perform up to 20 intravenous injections in mouse (50 µg of DNA per injection). Formation of complexes with the provided glucose solution produces nanoparticules adapted to in vivo injections. Bulk quantities are available upon request. |
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Please note that neither glucose solution nor DOPE are included. |
1. Hassani, Z., Lemkine, G.F., Erbacher, P., Palmier, K., Alfama, G., Giovannangeli, C., Behr, J.P. and Demeneix, B.A. (2005) Lipid-mediated siRNA delivery down-regulates exogenous gene expression in the mouse brain at picomolar levels. J Gene Med 7(2): 198-207.
2. Froidevaux M. S., Berg P., Seugnet I., Decherf S., Becker N., Sachs L. M., Bilesimo P., Nygard M., Pongratz I., Demeneix B. A., (2006) The co-chaperone XAP2 is required for activation of hypothalamic thyrotropin-releasing hormone transcription in vivo EMBO Rep (7) 1182.
3. Guissouma H., Froidevaux M. S., Hassani Z., Demeneix B. A., (2006) In vivo siRNA delivery to the mouse hypothalamus confirms distinct roles of TR beta isoforms in regulating TRH transcription Neurosci Lett (406) 240-3.
4. Kumar P., Lee S. K., Shankar P. and Manjunath N. (2006) A single siRNA suppresses fatal encephalllitis induced by two different flaviviruses PLOS Med 3: 0505-1.
Product SheetProtocol
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1 ml of FectoFly™ is sufficient to perform 100 to 200 transfections in 6-well plates. For bulk sizes, Please contact us. |
By simple and rapid transient transfection method, FectoFly™ provides high transfection efficiency in insect cells and robust protein production over several days (Fig. 1).
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Figure 2. Comparison of FectoFly™ in various adherent cell lines. Sf9, Sf21, S2 or Tn5 adherent cells were transfected in 24-well plates pCMV-EGFP-Luc plasmid. Luciferase assays were performed 72 h after transfection. |
FectoFly™ allows successful transfection of adherent and suspension insect cell cultures both in the latest generation of syntheticserum-free media (Fig.1, 3 – Sf21 and Sf9) and in the presence of serum (Fig.3 – S2). Transfection efficiency ranges routinely from 30% to 50% and cell viability is above 80%.
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Fig 3. GFP expression in Sf21, Sf9 cells grown in suspension, 72 h after transfection of pCMV-EGFP-Luc plasmid using FectoFly™. |
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FectoFly™ was compared to other commercially available reagents dedicated to the transfection of insect cells (Fig. 4). As shown for Sf9 cells, the highest protein expression levels were obtained with FectoFly™.
| Transfection of insect cells with FectoFly™ is straightforward. o Fast : 3 steps protocol o Simple: 1 µl of FectoFly™ per µg of DNA, 1:1 ratio o Versatile: efficient in serum-free synthetic media and in the presence of serum
Figure 5. FectoFly™ standard protocol. |
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Large volumes are available upon request |
Fecturin™ is ideal for the production of recombinant protein in HEK-293 and derivatives as well as in CHO cells grown either adherently or in suspension in synthetic media. This reagent is extremely gentle to cells, a strong advantage upon adaptation to suspension culture. This in turn increases protein yields dramatically. In addition, transfection efficiency with Fecturin™ ranges between 75 – 85 % (Fig. 1).
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Figure 1. Transfection of HEK-293 and CHO using Fecturin™. Cells were seeded in either FS or C-CHO media, two commercially available synthetic media. HEK-293 (left) or CHO (right) cells were transfected with pCMV-EGFP plasmid following the standard Fecturin™ protocol. Cells were visualized by fluorescent microscopy 24 h after transfection. |
In order to match stringent regulatory requirements in production laboratories, Fecturin™ is chemically defined and guaranteed free of any component of animal origin. Additional QC certificates may be provided upon request. Fecturin™ of various quality grades, including qualified for drug biomanufacturing, is also available upon request.
High levels of secreted VEGF (400 ng VEGF/106 cells/day) are obtained following transfection of HEK-293 with Fecturin™ in synthetic media. Moreover, Fecturin™ compares favourably to the commercially available reagent LP1 (Fig. 2).
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Figure 2. Compaison of protein expression levels after transfection of HEK293 cells with Fecturin™ and LP1 reagent. Suspension cells were seeded in FS medium and transfected with pCMV- Luc plasmid using Fecturin™ or LP1 following the manufacturer’s recommendations. Luciferase protein content is quantified 24 h post-transfection. |
Fecturin™mediated transfection results in high levels of protein production in CHO suspension culture when compared to LP2 lipid-based reagent, in two different commercially available synthetic media dedicated to CHO cells (Fig. 3). Up to 9 µg of luciferase/106 cells/ml/day is produced using Fecturin™ in C-CHO medium.
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Figure 3. Compaison of protein expression levels after transfection of suspension CHO cells with Fecturin™ and LP2 in two commercially available synthetic media. Cells were seeded in either I-CHO or C-CHO synthetic media and transfected with pCMV-Luc plasmid using either Fecturin™ or LP2 following the manufacturer’s recommendations. Luciferase expression (RLU) was assayed 24h after transfection. |
Upon transfection of CHO cells with Fecturin™, the daily expression of secreted VEGF (Vascular Endothelial Growth Factor) increases over four days (Fig. 4).
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Figure 4. Daily VEGF production in suspension CHO cells. Cells were seeded in C-CHO synthetic medium and transfected with pCMV-VEGF plasmid using Fecturin™ VEGF protein production was quantified by ELISA. |
The cumulative VEGF production also increases steadily over 9 days (Fig. 5).
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Figure 5. Cumulative VEGF production in CHO cells in suspension. Cells were seeded in C-CHO synthetic medium and transfected with pCMV-VEGF plasmid using Fecturin™. VEGF protein production was quantified by ELISA. |
Protein production in suspension culture of CHO cells can be easily scaled-up with Fecturin™. This reagent has been used to produce VEGF in 500 ml suspension culture of CHO cells with yields up to 10 µg of VEGF/106 cells /ml /day (Fig. 6). Protein production yield per ml increases significantly as the cell culture volume is scaled up. Protocols for 1 L or more are available upon request from our technical support.
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Figure 6. VEGF production in increasing volumes of culture. CHO cells were seeded in increasing volumes of C-CHO synthetic medium and transfected with pCMV-VECG plasmid using Fecturin™. Amount of VEGF protein produced was quantified by ELISA, 2 days after transfection. |
Increase the yield of recombinat proteins with Fecturin transfection reagent in serum free, suspension culture.
Polyplus News # 2 p. 6-9, 2006. (636 KB)
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Complexes formation is perfectly performed with this NaCl solution.0.5 ml of fluorescent jetPEI™ is sufficient to performed 250 transfections in 24-well plates. |
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Complexes formation is perfectly performed with this NaCl solution.0.5 ml of fluorescent jetPEI™ is sufficient to performed 250 transfections in 24-well plates. |
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0.4 ml of PULSin™ protein delivery reagent is sufficient for 24 reactions in 6-well plates. For bulk quantities, please contact us. |
PROTEINS
PULSin™ was shown to deliver R-phycoerythrin, a fluorescent protein (240 kD) to the cytoplasm of up to 98% cells. As shown in Figure 1, the protein is evenly distributed in the cytoplasm and excluded from the nucleus due to its large size.
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Fig 1. PULSin™-mediated intracellular delivery of R-phycoerythrin to NIH-3T3 cells. R-phycoerythrin (1 µg) was complexed with 4 µl of PULSin™ for 15 min and added to NIH-3T3 cells in a 24-well plate. Live cells were observed by fluorescence microscopy after 16 h. |
ANTIBODIES
Antibodies were also successfully delivered to HeLa cells and able to recognize their target protein inside the cytoplasm.
For example, PULSin™ permits the delivery of FITC-labeled anti-alpha-tubulin to the cytoplasm of 85% HeLa cells (Fig.2).
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Fig 2. Delivery of a fluorescein-conjugated anti-alpha-tubulin antibody with PULSin™ to HeLa cells. |
Similarly, anti-giantin Alexa Fluor® 488 was delivered to the cytoplasm of 98% of live HeLa cells, labeling the Golgi apparatus (Fig.3).
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Fig 3. Golgi labeling (green) of HeLa cells 24 h after delivery of 1 µg Alexa Fluor® 488 anti-Giantin using PULSin™. |
Plasma membrane was stained with ConA-rhodamine. Cells were observed by confocal microscopy.
PEPTIDES
Peptides are biomolecules acting with high specificity at low concentrations. The delivery of substrate, inhibitor, modulator, or blocking peptides into cell allows protein function studies as well as the development of therapeutic approaches. PULSin™ was shown to successfully deliver Streptococcus TPE B epitope into HeLa cells (Fig. 4).
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Fig 4. Delivery of Pep-A (Streptococcus TPE B epitope, 16 aa), into HeLa cells. Complexes were formed with Pep-A (1 µg, lissamine-rhodamine derivative, Sigma) and PULSin™ (4 µl). Observation was carried out 16 h post-delivery. |
Other proteins, antibodies and peptides have also been delivered to cells using PULSin™ (Table 1).
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Table 1. Examples of proteins, antibodies and peptides delivered to cells using PULSin™ |
| PULSin™ will save you time and efforts compared to other techniques using viral transduction or chemical conjugation.PULSin™ reagent is ready-to-use and provided with a dilution buffer and a fluorescent control protein (R-phycoerythrin).
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PULSin™ was shown to deliver proteins and antibodies to a large variety of cell lines and primary cells (Table 2, Fig. 1-3).
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Table 2. Efficiency of R-phycoerythrin delivery using PULSin™ in selected cells. |
The comparison of PULSin™ with two other protein delivery reagents showed a higher efficiency of protein delivery (Fig. 5). Moreover, the amount of protein delivered per cell was higher with PULSin™ as measured for R-phycoerythrin protein and for FITC-alpha-tubulin antibody (Fig. 6).
Fig 5. Comparison of PULSin™ efficiency with two other protein delivery reagents. R-phycoerythrin (1 µg) was complexed with each reagent according to the manufacturer’s protocol. Complexes were added to HeLa cells and observed by fluorescence microscopy over 24 hours.
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Fig 6. FACS analysis of HeLa cells after delivery of R-phycoerythrin (upper graph) or FITC-anti-alpha-tubulin antibody (lower graph) with PULSin™ or with competitor reagents B and C.Data are presented as histograms of the mean intensity of fluorescence for each cell population. |
PULSin™ contains a proprietary cationic amphiphile molecule that forms non-covalent complexes with proteins and antibodies.
Complexes are internalized via anionic cell-adhesion receptors and are released into the cytoplasm where they disassemble. The process is non-toxic and delivers functional proteins.
Weill, C., Biri, S., and Erbacher, P. (2008b). Cationic lipid-mediated intracellular delivery of antibodies into live cells. Biotechniques 44, Pvii-Pxi.7
Weill, C., Biri, S., Adib, A., and Erbacher, P. (2008a). A practical approach for intracellular protein delivery. Cytotechnology 56, 41-48.
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1 ml of FectoFly™ is sufficient to perform 100 to 200 transfections in 6-well plates. For bulk sizes, Please contact us. |
By simple and rapid transient transfection method, FectoFly™ provides high transfection efficiency in insect cells and robust protein production over several days (Fig. 1).
FectoFly™ allows successful transfection of adherent and suspension insect cell cultures both in the latest generation of syntheticserum-free media (Fig.1, 3 – Sf21 and Sf9) and in the presence of serum (Fig.3 – S2). Transfection efficiency ranges routinely from 30% to 50% and cell viability is above 80%.
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Fig 3. GFP expression in Sf21, Sf9 cells grown in suspension, 72 h after transfection of pCMV-EGFP-Luc plasmid using FectoFly™. |
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FectoFly™ was compared to other commercially available reagents dedicated to the transfection of insect cells (Fig. 4). As shown for Sf9 cells, the highest protein expression levels were obtained with FectoFly™.
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Transfection of insect cells with FectoFly™ is straightforward.
Figure 5. FectoFly™ standard protocol. |
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Complexes formation is perfectly performed with this NaCl solution.0.5 ml of jetPEI™-RGD is sufficient to perform 250 transfections in 24-well plates. |
jetPEI™-RGD/DNA complexes present many short RGD peptides on their surface and mimic the integrin’s natural ligands, hence the targeting. Integrin binding was shown to increase transfection of epithelial (HeLa) and fibroblast (MRC5) cells up to 100 fold in the presence of serum, when compared to jetPEI™. Enhancement is lost when aspartic acid is replaced by glutamic acid in the peptide sequence (RGD/RGE), thus confirming the involvement of integrins in the transfection pathway (Figure 1). Enhancement is lost when aspartic acid is replaced by glutamic acid in the peptide sequence (RGD/RGE), thus confirming the involvement of integrins in the transfection pathway .
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Figure 1. Transfection with RGD-conjugated PEI. Transfection of HeLa cells in serum-containing medium with a plasmid containing the firefly luciferase reporter gene (pCMVLuc) complexed with jetPEI™-RGD, or with PEI-RGE at N/P = 10. Luciferase activity was measured 24 h after transfection |
jetPEI™-RGD 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™-RGD is compatible with serum and antibiotics. No media changes or washes are required. Transgenic protein expression is determined 24 h to 72 h post-transfection.
1. Erbacher P., J. S. Remy and J. P. Behr (1999) Gene transfer with synthetic virus-like particles via the integrin- mediated endocytosis pathway. Gene Ther 6, 138-45
2. Pierschbacher M. D. and E. Ruoslathi (1984) Cell attachment activity of fibronectin can be duplicted by small fragments of the molecule. Nature 309, 30-33
3. Akiyama S. K. and K. M. Yamada (1985) Synthetic peptides competitively inhibit both direct binding to fibroblasts and functional biological assays for the purified cell-binding domain of fibronectin. J. Biol. Chem. 260, 10402-10405
4. Croyle M. A., E. Walter, S. Janich, B. J. Roessler and G. L. Amidon (1998) Role of integrin expression in adenovirus-mediated gene delivery to the intestinal epithelium. Hum Gene Ther 9, 561-573
5. Jackson T., A. Sharma, R. AbuGhazaleh, W. E. Blakemore, F. M. Ellard, D. L. Simmons, J. W. I. Newman, D. I. Stuart and A. M. Q. King (1997) Arginine-glycine aspartic acid-specific binding by foot-and-mouth disease viruses to the purified integrin alpha v beta 3 in vitro. J Virol 71, 8357-8361
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1.5 ml is sufficient to perform ca.375 transfections in 6-well plates.Bulk quantities are available upon request.Please contact us. |
Using jetPRIME™ reagent, DNA transfection efficiencies range between 70 and 90% on adherent cell lines, using only 2 µg of DNA and 4 µl of reagent per well in 6-well plates (Fig. 1).
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Fig. 1. GFP expression in HEK-293 and SK-OV-3 cells 24 h after transfection using 2 µg pCMV-GFP plasmid and 4 µl of jetPRIME™ in 6-well plates. |
jetPRIME™ is such a powerful in vitro transfection reagent that it only requires a small amount of plasmid DNA and reagent (Fig. 2). As a result, it is a very economical reagent.
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Fig. 2. Amounts of DNA and reagent (jetPRIME™ and competitor) added per well 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.
jetPRIME™ is extremely gentle to cells during transfection leading to increased cell viability (Fig. 3) and improved transfection results. Cells transfected with jetPRIME™ are healthy, while major cytotoxicity is observed with competitor.
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Fig. 3. Phase contrast microscopy of HeLa cells 24 h after transfections performed according to the manufacturer’s recommendations for each reagent. |
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. 4).
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Fig. 4. 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. |
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. 5).
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Fig. 5. Exogenous luciferase gene silencing in several cell lines after DNA & siRNA cotransfection using jetPRIME™ performed with 400 ng pCMV-Luc and 10 nM of luciferase siRNA per well in 6-well plates. |
jetPRIME™ is an easy-to-use transfection reagent (Fig. 6):
• Fast and easy to scale up and down
• Compatible with serum and antibiotics
Fig. 6. jetPRIME™ convenient protocol for DNA, siRNA and co-transfection of DNA and siRNA. |
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