Annual Report 2006

terminus, respectively. A signal sequence and a
trans-membrane sequence from either the
mouse gene or the mouse gene were
used. Some constructs contains the gene
sequence next to the gene
sequence. Totally eight kinds of fusion genes
were constructed in this study. To examine the
expression of the fusion genes on mammalian
cell surface, a series of plasmids encoding the
fusion protein were transfected into the HeLa
cells. Expression of the fusion protein on cell
surface was observed for any of the constructs.
Then, an antibody-mediated immunomagnetic
separation methodology was applied to
separate transformed cells. Transfected cells
were incubated with a polyclonal antibody
against streptavidin, and the antibody bound
cells were pulled out using a paramagnetic
beads coupled with the corresponding
secondary antibody. Highly pure population of
transformed cells was separated (Fig. 4). Then,
effects of fusion proteins on cell growth were
assayed. Cell proliferation rate of transformed
HeLa cells were compared with that of the
untransformed HeLa cells. No significant
difference of cell growth was observed in any of
the fusion genes. The property of eight fusion
genes developed in this study was found to be
similar. These results suggest that the fusion
genes and the immunomagnetic separation
protocol are useful for various transformation
applications.
Fig. 4
Expression of fusion protein on HeLa cells transfected with a plasmid encoding the FSAH2K-EGFP fusion protein
(A) Localization of streptavidin antigen on the surface of a transformed cell. Serial images captured at 5-µm
intervals on the z-axis.
(B) Transfected HeLa cells. Red staining indicates transformed cells.
(C-E) Immunomagnetically separated cells, same field.
(C) Light-interference view.
(D) Cells immunostained with anti-streptavidin antibody.
(E) EGFP-positive cells.
Scale bar: 10 mm (A) and 100 mm (B-E).