before cell confluence, the cultures are enriched for glial elements by exposure to serum free DMEM (47) 
for a period of 5 days with two medium changes to remove detached cells. The cells are then tranferred 
to 75 cm2 culture flasks (Gibco) for standard trypsinization methodology. 
Human Glioma Transfection Protocol We have described the 
transfection of rat glioblastoma cells by the episome based vector (30,34). Since these early animal 
studies, we have had experience in transfecting this episome into cultures derived from anaplastic 
human astrocytomas as well as glioblastomas. These tumors were obtained as discarded tissue from 
surgery at University Hospitals of Cleveland. After a week the cells are subcultured and transfected 
with the episome-based plasmid containing antisense to IGF-I. 
The construction of this plasmid was as previously described and as depicted in the following 
schematic. 
This antisense expression vector is episomal 
and includes the Epstein-Barr virus origin of 
replication and the gene encoding nuclear 
antigen I, which together drive 
extrachromosomal replication. The vector 
replicates readily within human cells 
providing a copy number of up to 100 
copies/cell(48). This allows for a high level of 
expression of anti-sense IGF-I RNA within the 
cells. The anti-sense IGF-I cDNA is linked to 
the highly inducible mouse metallo-thionein 
promotor. Downstream of the antisense DNA is 
a polyadenylation termination signal. The 
construct also contains genes for both 
hygromycin and ampicillin resistance. 
Cells are transfected using the Lipofectin reagent kit available from Gibco BRL according to the 
manufacturer's instructions. The method is based on encapsulation of the DNA within cationic 
liposomes which are then fused to the cells' surface. Essentially, 1-5 pg of DNA is mixed with the 
Lipofectin liposome suspension and applied to actively growing cells in low serum culture medium (Opti 
mem; Gibco-BRL). Following overnight incubation, the liposome-containing medium is aspirated and 
replaced with normal growth medium. After an additional 48 hours of incubation, hygromycin B is 
added at a concentration ( which must be established for each cell line) previously determined to kill 
untransfected cells over the course of about a week. The cultures are monitored until only actively 
growing colonies are present, at which time they are transferred to flasks and propagated in the 
continuous presence of hygromycin in order to maintain selective pressure. 
Using this technique, stable transfectants of the human glioblastoma or astrocytoma cells were 
derived. The addition of zinc sulfate to the culture media of the transfected cells activate the MT1 
promoter that drives the expression of the antisense IGF-I RNA. This expression was demonstrated by 
RNA transfer blot hybridization (Northern). The activation of this promoter by zinc sulfate resulted in 
Recombinant DNA Research, Volume 18 
[ 136 ] 
