C. HESDORFFER 4/93 
(32). In these studies, we have collaborated with 
Drs. Ira Pastan and Michael Gottesman (National Cancer Institute) 
in the use of an MDR cDNA in a retroviral vector constructed by 
these investigators (37) (Figure 1). This retroviral vector 
contains the entire coding sequence of the MDR gene and its 
expression driven by Harvey leukemia virus LTRs , and also 
contains a Harvey LTR enhancer. We have transfected this 
retroviral vector into our ecotropic and amphotropic packaging 
lines, and selected producer cells containing and expressing the 
MDR gene by exposure to colchicine (32,39). Cells that do not 
express the MDR gene are killed, while cells producing high 
levels of MDR survive. We have isolated several surviving clones 
and chosen those with the highest viral titers: 5 X 10 5 viral 
particles/ml for the ecotropic line, and 5 X 10* viral 
particles/ml for the amphotropic MDR producer line. We have used 
several transfect/infect protocols in an attempt to increase 
retroviral titers, but without success. In this scheme, we 
transfected the MDR-containing retroviral vector into our GP+E-86 
ecotropic packaging cells, and selected for colchicine-resistant 
clones. We then used viral supernatants from the highest titer 
ecotropic clone and infected amphotropic packaging cells. Clones 
were selected for colchicine resistance and titered on uninfected 
3T3 cells. 
We cocultured our highest titer (5 X 10 5 particles/ml) ecotropic 
producer clone with mouse bone marrow cells (32) , and transfused 
the transduced mouse bone marrow cells into lethally irradiated 
mice as described (32) . We demonstrated the presence of the 
human MDR gene in the peripheral blood of 90% of the transplanted 
mice using PCR analysis with human MDR-specific oligonucleotide 
primers (43) 50 days posttransplantation (32) . By contrast, no 
MDR signal is seen in the tail vein blood of untransplanted mice. 
In other experiments, we have examined the long-term expression 
of the human MDR gene in mice, and the cell types in which the 
inserted gene is expressed. At eight months posttransplantation, 
50% of the successfully transplanted mice continue to contain the 
human MDR gene by PCR of peripheral blood (32) . Southern blots 
of bone marrow from sacrificed animals demonstrate human MDR 
(90) . 
To analyze the expression of the human MDR gene at the protein 
level, we have used a monoclonal antibody, 17F9 (46), reacting 
with an external epitope of MDR p-glygoprotein on the surface of 
cells (provided by Dr. David Ring of Chiron Corporation) . Cells 
are first reacted with 17F9 and then exposed to a fluorescent 
anti-IgG2b (32) . Using 17F9 in fluorescent-activated cell 
sorting (FACS) analysis, we have shown that clones of both 
producer 3T3 cells and MELC containing the MDR cDNA retroviral 
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Recombinant DNA Research, Volume 18 
