C. HESDORFFER 4/93 
nucleated bone marrow cells (NBMC) from whole marrow for 24 hours 
with media containing 10% fetal calf serum, 10 units/ml IL-3, 200 
units/ml IL-6, and 50 units/ml human SCF (a gift from AMGEN) 
(107) . The NBMC were then exposed to A12M1 supernatants for 4-8 
hours, 3-4 X, over 48 hours to optimize retroviral transfer by 
providing an excess of virus over cells (usually 2-4 X 10 7 viral 
particles/10 7 cells) (107) . PCR analysis with MDR primers from 
different exons yields a unique 157 basepair band from transduced 
integrated MDR cDNA ; the endogenous human MDR gene containing 
introns gives either no signal or a band of larger size. A 
positive MDR PCR signal has been obtained after AM12M1 
supernatants are exposed to human bone marrow cells (107) 
(Figure 3). In addition, we have shown that increasing the 
number of times the supernatant is changed increases the PCR 
signal until excess virus is added (107) (Figure 1) . By analysis 
of BFU-E from transduced marrows, 10-50% of the colonies contain 
and express MDR as assayed by: 1) MDR PCR of individual 
colonies; and 2) Resistance of colonies to 60 ng/ml of 
colchicine, or both. In one experiment, 10% of BFU-E were MDR 
PCR-positive and colchicine-resistant (107) . In two other 
experiments, six of 12 and five of 12 individual BFU-E were MDR 
PCR-positive after transduction. We have also demonstrated that 
20-25% of human marrow cells transduced with MDR express the MDR 
protein at increased levels by FACS analysis (Figure 4). We have 
also used CD34+ cells isolated from CD34 antibody-coated plates 
(Applied Immune Sciences) , and can show MDR transduction of these 
cells as well (107) . 
GP+ env AM-12 packaging cells have been shown to be safe by their 
use with the IL-2 gene in human melanoma cells in culture (85) , 
an ADA gene in monkey bone marrow transfer experiments (108), and 
in our own studies with MDR of A12M1 cells (32,47). Reverse 
transcriptase assays, Mus dunni co-culture studies, and 
utilization of supernatants on naive 3T3 cells, have all been 
used in these experiments. The GP+envAM-12 packaging cells have 
been approved by the RAC and the FDA for use with the 11-2 gene 
to treat patients with malignant melanoma (85) . 
We have established a collaboration with Dr. John Dick 
(University of Toronto) , and currently are studying SCID mice 
infused with both untransduced and MDR-transduced human bone 
marrow cells using the protocol described earlier (107). In 
these experiments, 2-4 X 10 7 bone marrow cells have been given 
via tail vein to each mouse. This system should serve as a live 
animal model to determine the efficiency and potential toxicity 
of the transfer and expression of the MDR gene in human bone 
marrow cells prior to human trials. 
Recombinant DNA Research, Volume 18 
[103] 
