Autologous Bone Marrow Transplantation - Blog Science Connections

718 Electric Field-Mediated DMA Transfer
disposable plastic cuvette (Sarstedt, Princeton, NJ) shortened at the top by 1
cm with platinum foil electrodes (0.025 mm x 50 mm x 9 mm: ALFA,
Danvers, MA) mounted down the sides with epoxy glue. The distance
between the electrodes was approximately 4.5 mm. The 1SCO power supply
emits a high-energy pulse that decays exponentially within 1 jusec to an
oscillating tail of 20 /isec duration. Elimination of the tail abolished DNA
transfer, suggesting that both the high-voltage pulse and the low-energy tail
are required for electroporation. The electroporation of hematopoietic cells
is performed with the power supply typically set to deliver a pulse of 1.9 kV,
which is equivalent to a field strength of 3.8 kV/cm. Efficient DNA transfer
into lymphoid cells has also been observed with a commercial device (BTX
Transfector 800, Biotechnologies and Experimental Research Inc., San
Diego, CA), which delivers a square pulse in nonconductive media.
CHARACTERIZATION OF DNA TRANSFER
The characterization of DNA transfer by electroporation was studied with
a variety of lymphoid cell lines (3). We found that the level of transient gene
expression, determined as chloramphenicol acetyltransferase (CAT) activity,
as well as stable expression, expressed as the number of transfected cells,
increased with increasing DNA concentration in the medium. The data
suggested that the number of cells taking up DNA increased with increasing
DNA concentration.
An analysis of pools of stable transf ectants showed that the total amount
of DNA introduced into cells increased with the DNA concentration, but when
individual clones within the pool were analyzed, most were found to contain a
low gene-copy number despite transfection with high DNA concentrations.
These data may be explained by the presence of a subpopulation of cells
taking up large amounts of DNA, perhaps because of an intrinsic biologic
difference or as the result of heterogeneity in electric field strength.
Higher levels of gene expression were obtained when linear rather than
supercoiled DNA was used for electroporation. Both transient and stable
gene expression were similarly increased, suggesting that the mechanism of
DNA transfer may be affected.
An analysis of isolated clones showed that the transferred sequence
integrated at random sites in the DNA and that unintegrated DNA was not
detected, even when electroporation was performed with high DNA concentrations.
We also found that integration occurred exclusively at the site of
linearization, so that the structure of the integrated DNA can be predicted.
We also observed that co-transfection of unlinked sequences proceeded
efficiently, since 50% to 100% of transformants were co-transformants.
Our attempts to improve transfection frequency by treating cells with
chloroquine or sodium butyrate, which have been reported to increase
transfection frequency by the DEAE dextran and calcium phosphate