Team:Westminster/Modeling

From 2012.igem.org

(Difference between revisions)
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<body>
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<div id="wrapper">
<div id="header">
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<div id="logo">
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  <div id="control-btn" class="btn-cog"></div>
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<a href="http://2012.igem.org/Team:Westminster">
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<img src="http://2012.igem.org/wiki/images/1/1e/Westminster-logo.png" alt="iSTEM" title="Back to home!" />
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<li><a href="http://2012.igem.org/Team:Westminster" class="navlink">Home</a></li>
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<li><a href="http://2012.igem.org/Team:Westminster/Team" class="navlink">Team</a></li>
<li><a href="http://2012.igem.org/Team:Westminster/Team" class="navlink">Team</a></li>
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<li><a href="http://igem.org/Team.cgi?year=2012&team_name=Westminster"class="navlink">Team Profile</a></li>
 
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<a href="http://2012.igem.org/Team:Westminster/Project/Overview"class="navlink">Project</a>
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<a href="http://2012.igem.org/Team:Westminster/Overview"class="navlink">Project</a>
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<li class="submenu-button"><a href="http://2012.igem.org/Team:Westminster/Overview"class="navlink">Overview</a></li>
<li class="submenu-button"><a href="http://2012.igem.org/Team:Westminster/Problem"class="navlink">The Problem</a></li>
<li class="submenu-button"><a href="http://2012.igem.org/Team:Westminster/Problem"class="navlink">The Problem</a></li>
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<li class="submenu-button"><a href="http://2012.igem.org/Team:Westminster/Safety"class="navlink">Safety</a></li>
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<li><a href="http://2012.igem.org/Team:Westminster/Parts"class="navlink">Parts</a></li>
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<li><a href="http://2012.igem.org/Team:Westminster/Modeling"class="navlink active">Modeling</a></li>
-
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<li><a href="http://2012.igem.org/Team:Westminster/Outreach"class="navlink">Outreach</a></li>
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<li><a href="http://2012.igem.org/Team:Westminster/Safety"class="navlink">Safety</a></li>
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                    <li><a href="http://2012.igem.org/Team:Westminster/Judging"class="navlink">Judging Criteria</a></li>
 +
 
 +
<li><a href="http://2012.igem.org/Team:Westminster/Attributions"class="navlink">Attributions</a></li>
<li><a href="http://2012.igem.org/Team:Westminster/Attributions"class="navlink">Attributions</a></li>
</ul><!-- #navigation-list -->
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</div><!-- #navigation -->
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<a href="http://2012.igem.org/Team:Westminster">
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<img src="http://2012.igem.org/wiki/images/a/ae/Logo-white.png" alt="iSTEM - intelligent synthetic tumour eliminating machine" width="172" height="66" title="iSTEM - intelligent synthetic tumour eliminating machine" />
 +
</a>
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</div><!-- #logo -->
</div><!-- #header -->
</div><!-- #header -->
<div id="the-content">
<div id="the-content">
-
<div id="home-main-image">
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<!--div id="home-main-image">
<img src="http://2012.igem.org/wiki/images/4/4d/Westminster_team.png" alt="Welcome" title="Welcome to our home!" />
<img src="http://2012.igem.org/wiki/images/4/4d/Westminster_team.png" alt="Welcome" title="Welcome to our home!" />
-
</div><!-- #home-main-image -->
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</div--><!-- #home-main-image -->
<div id="home-main-text">
<div id="home-main-text">
-
<h2>Lorem Ipsum</h2>
+
-
<p>Cancer recurrence is one of the fears that almost every patient undergoing chemotherapy develops. Recent findings suggest that only a small fraction of the tumor cells, called Cancer Stem Cells (CSC) are able to drive the growth of the tumor. CSC also show an increased drug resistance, and could remain unaffected after chemotherapy, eventually resulting in the formation of a new tumor.</p>
+
  <p><b>Modelling of PEI transfection</b></p>
-
<div class="centered"><img src="http://2012.igem.org/wiki/images/4/4d/Westminster_team.png" /></div>
+
  <p><span style='color:red'>What is
-
<h2>Lorem Ipsum</h2>
+
    PEI?</span></p>
-
<p>Cancer recurrence is one of the fears that almost every patient undergoing chemotherapy develops. Recent findings suggest that only a small fraction of the tumor cells, called Cancer Stem Cells (CSC) are able to drive the growth of the tumor. CSC also show an increased drug resistance, and could remain unaffected after chemotherapy, eventually resulting in the formation of a new tumor.</p>
+
  <p>Poly (<span class=SpellE>ethylalenimine</span>) was
-
<div class="centered"><img src="http://2012.igem.org/wiki/images/4/4d/Westminster_team.png" /></div>
+
    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. </p>
 +
  <p><span style='color:red'>How is the DNA released in the cell?</span></p>
 +
  <p>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. </p>
 +
  <p><span style='color:red'>Why isn’t PEI more widely used?</span></p>
 +
  <p style='line-height:14.4pt;background:white'><span style='font-size:10.0pt;
 +
font-family:Arial;color:black'>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. </span></p>
 +
  <p style='line-height:14.4pt;background:white'><span style='font-size:10.0pt;
 +
font-family:Arial;color:red'>If it is toxic to cells, why suggest <span
 +
class=GramE>it’s</span> use?</span></p>
 +
  <p style='line-height:14.4pt;background:white'><span style='font-size:10.0pt;
 +
font-family:Arial;color:black'>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! &nbsp;</span></p>
 +
  <p><span style='color:red'>Protocol for preparing the PEI-DNA
 +
    mixture and transfection of MCF7 cells (As performed by Andrew Jenks)</span></p>
 +
  <p>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. </p>
 +
  <p>Procedure </p>
 +
  <p>On the day prior to transfection 250000 MCF7 cells were
 +
    seeded per well of a 24 well plate.</p>
 +
  <p>For each transfection, reagents were prepared as follows:</p>
 +
  <p class=MsoListParagraphCxSpFirst><span
 +
lang=EN-US style='font-family:Calibri;'>a)<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span lang=EN-US style='font-family:Calibri;
 +
'>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. </span></p>
 +
  <p class=MsoListParagraphCxSpMiddle><span
 +
lang=EN-US style='font-family:Calibri;'>b)<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span lang=EN-US style='font-family:Calibri;
 +
'>100 µl (10% of growth media volume) of
 +
    0.15M <span class=SpellE>NaCl</span> was added to plasmid-PEI mixture</span></p>
 +
  <p class=MsoListParagraphCxSpMiddle><span
 +
lang=EN-US style='font-family:Calibri;'>c)<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span lang=EN-US style='font-family:Calibri;
 +
'>Transfection reagent mixture was <span
 +
class=SpellE>vortexed</span> and incubated at room temperature for 10 <span
 +
class=SpellE>mins</span>.</span></p>
 +
  <p>The 24 well <span class=GramE>plate</span> 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-<span class=SpellE>NaCl</span> mixture was the added <span class=SpellE>dropwise</span> with even distribution
 +
    to cells. Cells were incubated overnight at 37&#7506;C. The following day, cells were assessed for cell health
 +
    and the media replaced. After this the cells were allowed to recover for <span
 +
class=GramE>48hrs&nbsp; before</span> being <span class=SpellE>trypsinised</span>. </p>
 +
  <p>Cells were analysed by flow <span class=SpellE>cytometery</span> as follows:</p>
 +
  <p>Media was removed and the cells were washed once with PBS.
 +
    Cells were then <span class=SpellE>trypsinised</span> with 0.21mM trypsin
 +
    containing 4.81 <span class=SpellE>mM</span> EDTA. The trypsin was then neutralized
 +
    with the addition of complete media and cells were centrifuged at 2000 rpm for
 +
    3 <span class=SpellE>mins</span></p>
 +
  <p>Cells were then washed once in ice cold PBS containing 1% foetal
 +
    bovine serum (FBS) <span class=GramE>and &nbsp;re</span>-suspended in 1% FBS containing
 +
    1ug/ml <span class=SpellE>propidium</span> iodide.</p>
 +
  <p>Cells were analysed using <span class=SpellE>CyAn</span>™
 +
    ADP flow cytometer (<span class=SpellE>DakoCytomation</span>). In order
 +
    to distinguish between alive and dead cells <span class=SpellE>propidum</span> 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.</p>
 +
  <p style='line-height:normal;'><b><span style='font-size:10.0pt;
 +
font-family:Arial;color:red;
 +
'>RESULTS:</span></b></p>
 +
  <p style='line-height:normal'><span style='font-size:10.0pt;font-family:Arial;"Times New Roman";color:black;'>Results of the transfection using the PEI and plasmid concentrations was
 +
    as follows:</span></p>
 +
  <p style='line-height:normal'><b><span lang=EN-US
 +
style='font-size:10.0pt;font-family:Arial;
 +
color:black;'><img width=384 height=227
 +
src="http://2012.igem.org/wiki/images/6/6f/Modelling_002.png" v:shapes="Picture_x0020_3"></span></b></p>
 +
  <p>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. </p>
 +
  <p><span lang=PT>Boussif, O et al.
 +
    (1995) A versatile vector for gene and oligonucleotide transfer into cells in
 +
    culture and in vivo: Polyethylenimine </span><a
 +
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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Revision as of 21:00, 26 September 2012

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.

Boussif, O et al. (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine http://www.pnas.org/content/92/16/7297.full.pdf+html