Team:Westminster/Modeling

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<img src="http://2012.igem.org/wiki/images/b/b1/Transfection_wmin.png" alt="transfections" title="transfections" width="800" height="479" />
<img src="http://2012.igem.org/wiki/images/b/b1/Transfection_wmin.png" alt="transfections" title="transfections" width="800" height="479" />
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   <p><span lang=PT>Boussif, O et al.
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   <p><span lang=PT>References:
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    (1995) A versatile vector for gene and oligonucleotide transfer into cells in
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Boussif, O et al. (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine. National Acad Science, 92 (16).
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    culture and in vivo: Polyethylenimine </span><a
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Akin, A. et al. (2004). Exploring polyethylenimine-mediated DNA transfection and the proton sponge hypothesis. The Journal of gene medicine, 7 (5).  
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href="http://www.pnas.org/content/92/16/7297.full.pdf+html"><span lang=PT>http://www.pnas.org/content/92/16/7297.full.pdf+html</span></a></p>
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Werth, S (2006). A low molecular weight fraction of polyethylenimine (PEI) displays increased transfection efficiency of DNA and siRNA in fresh or lyophilized complexes. Journal of Controlled Release, 11 (2) 257-270.
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Revision as of 01:49, 27 September 2012

Welcome

Modelling of PEI transfection

What is PEI?

Poly (ethylalenimine) was identified as a transfection reagent in 1995. It is a linear cationic polymer which condenses negatively charged DNA molecules to produce positively charged particles. These positively charged complexes interact with negatively charged cell surface residues and are internalised via endocytosis.

How is the DNA released in the cell?

Once inside the cell, the amine groups of the PEI polymer accept protons to become more positively charged. This causes an influx of counter ions resulting in osmotic swelling. Osmotic swelling leads to bursting of the vesicle, and release of the DNA-PEI complex. The PEI unpacks from the DNA in the cytoplasm allowing the DNA to diffuse to the nucleus.

Why isn’t PEI more widely used?

PEI is extremely cytotoxic. It causes disruption of the cell membrane which then leads to immediate cell death or it may disrupts the mitochondrial membrane which leads to delayed death of cells.

If it is toxic to cells, why suggest it’s use?

The high cost of mammalian cell culture is possibly one of the restricting factors encouraging iGEM teams and other researchers from working with mammalian cells. PEI is a very cheap transfection reagent, which works!  

Protocol for preparing the PEI-DNA mixture and transfection of MCF7 cells (As performed by Andrew Jenks)

All cell culture and transfection was carried in complete media Dulbecco’s modified eagles media (DMEM) supplemented with 10 % foetal bovine serum (FBS). No antibiotics were used, cells were transiently transfected.

Procedure

On the day prior to transfection 250000 MCF7 cells were seeded per well of a 24 well plate.

For each transfection, reagents were prepared as follows:

a)     Plasmid mixed with PEI.PEI volumes used were either 0 µl, 3 µl, 6 µl, 9 µl, 12 µl and 15 µl. Plasmid concentration was either at 0 µg, 0.36 µg, 0.7 µg, 1.46 µg, 2.92 µg and 5.84 µg. Combinations of the PEI and plasmid was used.

b)     100 µl (10% of growth media volume) of 0.15M NaCl was added to plasmid-PEI mixture

c)      Transfection reagent mixture was vortexed and incubated at room temperature for 10 mins.

The 24 well plate was removed from the incubator. Prior to transfection, old media was removed from each well and replaced with fresh media. Prior to transfection, the old media from each well was replaced with fresh media. The PEI-plasmid-NaCl mixture was the added dropwise with even distribution to cells. Cells were incubated overnight at 37ᵒC. The following day, cells were assessed for cell health and the media replaced. After this the cells were allowed to recover for 48hrs  before being trypsinised.

Cells were analysed by flow cytometery as follows:

Media was removed and the cells were washed once with PBS. Cells were then trypsinised with 0.21mM trypsin containing 4.81 mM EDTA. The trypsin was then neutralized with the addition of complete media and cells were centrifuged at 2000 rpm for 3 mins

Cells were then washed once in ice cold PBS containing 1% foetal bovine serum (FBS) and  re-suspended in 1% FBS containing 1ug/ml propidium iodide.

Cells were analysed using CyAn™ ADP flow cytometer (DakoCytomation). In order to distinguish between alive and dead cells propidum iodide was used and data was analysed using the summit v4.3 software. Cell lines were gated according to an unstained sample and lasers were adjusted accordingly. Unstained cells were used to set gates for cell size and internal complexity, dead cells were removed along with doublets.

RESULTS:

Results of the transfection using the PEI and plasmid concentrations was as follows:

6 µl of PEI with 1.46 µg of plasmid gave the highest level of transfection. 43% of cells were transfected. This is highly favourable in comparison to other transfection reagents. Increasing the DNA concentration did not improve transfection.

transfections

References: Boussif, O et al. (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine. National Acad Science, 92 (16). Akin, A. et al. (2004). Exploring polyethylenimine-mediated DNA transfection and the proton sponge hypothesis. The Journal of gene medicine, 7 (5). Werth, S (2006). A low molecular weight fraction of polyethylenimine (PEI) displays increased transfection efficiency of DNA and siRNA in fresh or lyophilized complexes. Journal of Controlled Release, 11 (2) 257-270.