Figure C. FACS analysis of human melanoma cells co-transfected 
with vector II and RSV-neo, followed by 6418 selection. Cells 
were incubated with monoclonal antibody to HLA-B7 or vWF 
(control) (left) . Cells were further enriched by FACS sorting 
and incubated with the same antibodies (right) . 
III. Mechanism of liposome-mediated gene transfer and 
integration status. 
Despite the fact that cationic liposomes have been used for 
several years to deliver recombinant genes to cells, the 
mechanism of gene transfer is not entirely clear. Although early 
studies suggested that DNA is delivered to cells through lipid- 
mediated membrane fusion, more recent data, obtained by electron 
microscopy and with functional studies, show that this process 
may be endosome-mediated (L. Huang, personal communication) . For 
the proposed studies, we have begun to examine stable vs. 
transient expression after liposomal transduction. By p- 
galactosidase staining, most expression is transient (> 95%) and 
episomal DNA is easily recovered in Hirt supernatants two days 
after transfection (2000-6000 colonies//zg of Hirt DNA) . When DNA 
is linearized by restriction enzyme digest prior to lipofection, 
a large proportion of expression remains transient; however, the 
numbers of stable lines isolated increases by a factor of -10- 
fold. Episomal DNA therefore appears to be lost as cells divide 
in culture if plasmid is not linearized. In non-dividing cells, 
episomal DNA and expression can be maintained for longer time 
periods. Within tumors in vivo, expression appears to be largely 
[432] 
Recombinant DNA Research, Volume 15 
