The main focus in proteomic studies is on the identification of proteins in given biological samples. Proteins isolated and separated from a given sample (e.g., whole cell lysates, blood or tissue, protein complexes) by immunoprecipitation or affinit more
Fugene® HD Transfection Reagent: Superior Performance for Challenging Expression StudiesMaria Manifava*
Department of Signalling, Babraham Institute, Cambridge, UK
The ability to efficiently introduce foreign molecules into cells constitutes an important tool in any laboratory and facilitates studies of gene regulation and protein function.
A plethora of transfection methods have been developed for transfer of DNA into mammalian and animal cells. Those methods either utilize the physicochemical properties of the vector-carrier of the DNA or use mammalian viruses. In the first group of methods, some classic techniques are well-known, such as: calcium phosphate , DEAE-dextran , electroporation [3, 4] liposome-mediated delivery , direct microinjection/biolistic particle delivery [6, 7], and non-liposomal multi-component reagents. Viral vector systems use both DNA (Adeno-) and RNA (Retro-, Lenti-) viruses.
None of these transfection methods is considered ideal since they all have advantages but pose limitations as well. A “perfect” method would potentially be the one that fulfils these requirements: high transfection efficiency, minimal cytotoxic effects/low cell mortality, high reproducibility, ability to transfect both transiently and stably a widespread spectrum of cell types, ease and low cost of the application.
In our lab, through the years we have used most of the aforementioned transfection methods. We have tested a number of lipid-based reagents and found that Fugene® 6 Transfection Reagent has performed for us more reliably than any other, using a wide range of cells types and DNA reporters. Recently we have been testing the newer version of Fugene® 6 Transfection Reagent, Fugene® HD Transfection Reagent. We have used mammalian and insect cell lines, with two kinds of DNA reporters, an ‘easy’ and a ‘difficult’ one. Fugene® HD Transfection Reagent has proven to be a very powerful reagent, giving higher transfection efficiencies, especially with difficult-to-express DNA reporters.
Materials and Methods
HEK-293 cells: Human embryonic kidney cells were grown in DMEM supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere of 95% air and 5% CO2. PAE cells: Porcine aortic endothelial cells were grown in F12-HAM supplemented with 10% heat-inactivated serum, and maintained in the same conditions as the HEK-293 cells. Drosophila S2 Schneider cells: The cells were grown in Shields and Sang M3 Insect medium supplemented with 10% heat-inactivated serum and 1% antibiotic-antimycotic, and maintained at 25°C.
The mammalian cells were subcultured the day before transfection onto glass coverslips. The day of the transfection the cell confluency was about 50%. The S2 cells were subcultured on 6-well multiplates the day of the transfection at 90% confluency.
Complexes were formed using either Fugene® HD Transfection Reagent or Fugene® 6 Transfection Reagent with a series of different constructs: pCMV3 GFP PLD1 , pEGFPC2 GFPSK1 , pRMHA3 myc-tagged Drosophila, PLD (DPLD, unpublished). The ratios of lipid to DNA varied from 3:1 (for the GFPSK1) to 3:2 (GFPPLD) up to 6:3 for the DPLD. The complex formation was left for 18 minutes (Fugene® HD Transfection Reagent) or 40 minutes (Fugene® 6 Transfection Reagent). The complex was formed in serum-free medium (OPTI MEM for Fugene® HD Transfection Reagent, or DMEM supplemented with 10 mM HEPES-NaOH PH 7.2 for Fugene® 6 Transfection Reagent). After formation, the complexes were dispersed onto the cells. Expression times ranged from 22 hours (GFP SK1) to 30 hours (GFP PLD) whereas for DPLD the expression was left for 42 hours including 24 hours induction of the metallotheinin promoter with 650 µM CuSO4. Before fixation and staining, the S2 cells were plated on glass coverslips coated with concavalin A for 1 hour to spread out.
Cells were washed in PBS and then fixed using 3.7% formaldehyde in 200 mm HEPES, pH 7.2. The cells were washed in DMEM to quench any remaining formaldehyde. The samples that required staining were permeabilized in NET GEL (150 mM NaCl, 5 mM EDTA, 50 mM Tris-Cl pH 7.4, 0.05% NP-40, 0.25% gelatine, 0.02% sodium azide) containing additionally 0.25% NP-40. Mouse hybridoma myc antibody was used to detect the myc-tagged DPLD. Finally, the coverslips were washed in deionised water and mounted on glass slides using Aqua Polymount. The samples were visualised using a Zeiss Axiophot and images were obtained using a SPOT digital camera.
Results and Discussion
Transfection of mammalian cells
To test directly the efficiency of Fugene® HD Transfection Reagent, we transfected several different mammalian cell lines. HEK-293 cells were our first choice since we use them routinely and they are a reliable biochemical machinery which supports the expression of a wide range of proteins from both mammalian and non-mammalian genes . In this case GFPPLD1 was transfected using the conditions referred to earlier. As a control, duplicate cells were transfected with Fugene® 6 Transfection Reagent, using conditions that have successfully given us, through the years, good protein expression in a variety of cell types (COS7, CHO, HEK, RBL, RAW264.7, PAE) and with a variety of plasmids. We evaluated the expression levels by immunofluoresence. As shown in Figure 1 many more cells express this construct with Fugene® HD Transfection Reagent in comparison to Fugene® 6 Transfection Reagent. A phase contrast depiction of those two fields (Figure 1, lower panel) shows similar cell numbers in each field. The same transfection conditions were used for another cell type, PAE. Again, as can be seen in Figure 1, the expression using Fugene® HD Transfection Reagent is far greater than for Fugene® 6 Transfection Reagent. While the efficiency with the former is well above 50%, with the latter it is almost nondetectable. Both of these cell lines were also transfected using the two re-agents with a GFP-SK1 construct and both gave very high transfection efficiencies (data not shown). We know from previous experience that any PLD-related construct is considered a difficult expressor, whereas SK1 constructs usually express better.
We concluded from these results that, whereas both Fugene® 6 Transfection Reagent and Fugene® HD Transfection Reagent are useful, the newer Fugene® HD reagent is superior for challenging expression studies. This is also true when comparing Fugene® HD Transfection Reagent with other high-efficiency transfection reagents such as transfection reagent L. Fugene® HD Transfection Reagent and transfection reagent L give similar (high) efficiencies for difficult constructs, but the advantage of Fugene® HD Transfection Reagent is that, in our hands, it causes very little cell death and is not as sensitive to initial cell density as the transfection reagent L.
Transfection of S2 cells
S2 Schneider cells were plated on 6 wells. Three types of complexes were prepared in both transfection reagents, using the same ratio of Fugene® Transfection Reagent: DNA with different serum-free media. The Fugene® Transfection Reagent:DNA ratio that was finally useful was 6:2. The different serum-free media that were check-ed were the Shields and Sang M3, the Drosophila SFM, and the OPTIMEM. The complexes were dispersed onto the cells within an hour of plating the cells. We observed that when OPTIMEM was used, we obtained the highest expression for both reagents. As shown in Figure 1, the transfection efficiency when using Fugene® HD Transfection Reagent was at least double in comparison to Fugene® 6 Transfection Reagent. The efficiency was evaluated using a difficult expressor (DPLD). This verifies our observation from the transfection on mammalian cell lines, that Fugene® HD Transfection Reagent is superior to Fugene® 6 Transfection Reagent.
It’s worth mentioning that in none of the above experiments were signs of cell cytotoxicity observed. For the mammalian cell lines the seeding confluencies varied from 30% to 60%, while for the Drosophila cell line the confluency varied from 60% to 90% (data not shown).
Fugene® HD Transfection Reagent is a very gentle but effective transfection reagent. It combines two extremely useful properties: high transfection efficiency for a broad range of cell types without causing cell death, even at confluencies of less than 50%. It is especially useful for difficult expressors and we have recently started using it effectively for plasmid-based RNAi work.
Fugene® HD Transfection Reagent is superior to Fugene® 6 Transfection Reagent in the described cases. However, Fugene® 6 Transfection Reagent is well-suit-ed to transfect a wide variety of cell lines not yet tested with Fugene® HD Transfection Reagent.
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3. Wong TK, Neumann E (1982) Biochem Biophys Res 107: 584–587
4. Shikegawa K, Dower WJ (1988) Biotechniques 6: 742–751
5. Felgner PL et al. (1987) Proc Natl Acad Sci USA 84: 7413–7417
6. Capecchi MR (1980) Cell 22: 479–488
7. Klein TM et al. (1987) Nature 327: 70–73
8. Corrotte M et al. (2006) Traffic 7: 365–377
9. Delon C et al. (2004) J Biol Chem 279: 44763–44774
10. Thomas P, Smart TG (2005) J Pharmacol Toxicol Methods 51: 187–200
This work was supported by the Biotechnology and Biological Sciences Research Council.
FUGENE is a registered trademark of Fugent L.L.C., USA.
This article was originally published in Biochemica 3/2006, pages 26-27. ©Springer Medizin Verlag 2006
- cell lines
- viral vectors
- serum-free media
- protein expression
- gene regulation
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