Autologous Bone Marrow Transplantation - Blog Science Connections

Electric Field-Mediated DNA Transfer 719
methods, respectively (10,11), were unsuccessful. The lack of success with
chloroquine is consistent with the notion that DNA introduced by electroporation
is not incorporated into lysosomes (12). Pretreatment with a mitoticarrest
agent such as Colcemid produced a twofold increase in transient gene
expression but did not influence stable expression.
The influence of different regulatory sequences on the transient
expression of DNA introduced by electroporation was also examined. In all
the lymphoid lines tested, the simian virus 40 early region was a better
promoter than was the Rous sarcoma virus long terminal repeat.
ELECTROPORATION OF HUMAN HEMATOPOIETIC
PROGENITOR CELLS
We have applied the electroporation method to the transfer of DNA into
human hematopoietic cells (4). The results of transient gene expression
studies demonstrated that normal human nucleated marrow cells were
successfully transfected by electroporation.
We next showed excellent human granulopoietic progenitor-cell recovery
(as measured in the colony-forming unit granulocyte-macrophage [CFCJ-
GM] assay) after exposure of nucleated marrow cells to the electric field
alone (96 ± 3%, n = 3). The granulocyte-macrophage colonies obtained after
electroporation were indistinguishable from control colonies. Electroporation
per se, therefore, does not affect human CFO-GM viability and probably
does not perturb hematopoiesis. We subsequently showed that selectable
genes (neo R , gtp) introduced into marrow cells by electroporation were
expressed in the progeny of hematopoietic progenitor cells. We found that
0.8% to 2.7% of CFCi-GM expressed the resistant phenotype. DNA transfer
was confirmed by hybridization analysis. We further concluded that this
transfection frequency may be a minimal value reflecting our highly stringent
selection conditions and may reflect a subpopulation of cells expressing high
levels of the transferred gene. The actual transfection frequency may be up to
15-fold higher.
Further improvements in transfection frequency may be achieved with
refinements in the electroporation apparatus and by more detailed analysis of
physical parameters, such as pulse shape, width, and frequency.
ADVANTAGES OF THE
ELECTROPORATION TECHNIQUE
Our studies of electric field-mediated DNA transfer have enabled us to
identify numerous advantages associated with the technique. They can be
summarized as follows: