Biomedical Engineering Reference
In-Depth Information
3.6 Preparation
of Cell Lysate
for Ultracentrifugation
in Iodixanol Gradient
1. 48 h after transfection aspirate all but 3-5 ml medium from
each dish, scrape the cells off using a cell scraper, and transfer
the media/cells into 50 ml Falcon tubes. Wash the dishes with
10-15 ml complete DMEM or PBS (starting from dish No. 1
and then transferring the medium to the next dish till all dishes
are washed) to harvest all the cells.
2. Centrifuge at 1,200 × g for 10 min at room temperature and
aspirate supernatant.
3. Resuspend cell pellets in 15 ml Lysis buffer (150 mM NaCl,
50 mM Tris-HCl, pH 8.5) per 10 dishes.
4. Freeze/thaw three times by alternate immersion in dry ice/
ethanol bath and 37 °C water bath.
5. Add Benzonase nuclease to the lysate to a final concentration
of 50 U/ml (750 U/15 ml). Incubate at 37 °C for 30-60 min.
6. Centrifuge at 4,000 ×
g
for 20 min and transfer the vector-
containing supernatant to a clean tube. It may be used imme-
diately or stored at 4 °C for several hours while the iodixanol
gradients are prepared.
7. Proceed to Sect. 3.8 for description of the tube loading, ultra-
centrifugation, and subsequent steps. Note that a single
Quick-Seal 39 ml centrifugation tube (Beckman Coulter
#344326) can accommodate up to 15 ml of lysate, and the
lysate from no more than 15 dishes should be loaded into a
single tube. More concentrated material would compromise
the resolution and the purity of the vector.
3.7 Polyethyenimine
(PEI) Transfection
As an alternative to calcium phosphate, polyethyenimine (PEI) can
be used to condense DNA and act as a carrier into targeted cells.
PEI condenses DNA into positively charged particles, which bind to
anionic cell surface residues and are brought into the cell via endo-
cytosis. Once inside the cell its relatively strong positive charge com-
bined with the acidification of the endosome results in an influx of
counter-ions and a lowering of the osmotic pressure. Osmotic swell-
ing occurs and the resulting osmotic gradient across the endosome's
membrane leads to an influx of water, bursting the endosome and
allowing the PEI and bound DNA (polyplex) to escape to the cyto-
plasm. If the polyplex unpacks, then the DNA is free to diffuse to
the nucleus. Further details of the DNA's transit to the nucleus are
not well established, but the extensive use has shown that PEI is an
efficient transfection reagent, yielding transfection efficiencies and
viral titers similar to the calcium phosphate precipitation method.
High charge density in physiological media and high buffer
capacity in weakly acidic media account for PEI's efficiency.
Though low molecular weight PEIs are less toxic, their gene trans-
fection efficiency is much lower than high molecular analogs such
as 25 kDa PEI. The gene transfer efficiency of PEI is attributed to
Search WWH ::
Custom Search