Transduction methods.

Solution DNA PCR analysis of vector MDR-1 positive cells. The cells from the suspension transduction protocol were incubated for an additional 24 hr in the presence of IL-3 and IL-6 growth factors in suspension culture after 6 hr of incubation with the vector before PCR assay. Cells that were subjected to the stromal or the suspension transduction procedures were cultured for an additional 24 hr after exposure to the retroviral vector in long-term culture on stromal monolayers before being collected for PCR assay. Cells were also obtained from the peripheral blood or bone marrow of patients at various times after transplant. DNA solution PCR analysis of vector MDR-1 sequences in the cells transduced with a MDR-1 retroviral vector. (A) DNA solution PCR analysis of vector MDR-1 sequences; (B) DNA solution PCR analysis of endogenous MDR-1 sequences. Lanes: 1, Phi-X174 molecular weight markers; 2, PCR buffer negative control; 3 and 4, marrow cells from ovarian cancer patient no. 9 unexposed and exposed to the MDR-1 vector suspension transduction method (both negative for the vector sequences); 5 and 6, nonadherent and adherent CD34⁺ cells from stromal cultures of breast cancer patient no. 5 not exposed to the MDR-1 vector (negative for MDR-1 vector sequences); 7–9, nonadherent hematopoietic cells, mixture of adherent hematopoietic cells and stromal cells, and stromal cells from cultures used for transduction with the MDR-1 vector stromal transduction method from breast cancer patient no. 5, respectively (all three samples positive for the vector MDR-1 sequences); 10, positive control consisting of cDNA from K562 cells known to contain the MDR-1 vector sequences.

In situ DNA PCR analysis of marrow or peripheral blood cells 24 hr posttransduction or from patients at various time intervals posttransplant. A Boehringer Mannheim kit was used for an in situ DNA PCR assay as described previously (16). The nuclei of the cells that are transduced turn blue, whereas those that are not transduced stay white. Controls that were run with every patient sample included cells known to be negative for the vector pVMDR-1 and the patient’s own pretransduction cells. The percent of cells positive in these negative controls (which is probably due to a primer independent repair incorporation reaction) was subtracted from the percent of cells positive for the blue intranuclear color in known negative cells (16). In situ DNA PCR analysis of negative control cells (A) and cells transduced by the stromal transduction method (B). The positive cells contain intensely staining blue nuclei.

DNA solution PCR analysis of marrow cells from patients transplanted with cells exposed to the stromal transduction method. Lanes: 1, Phi-X174 molecular weight markers; 2, negative buffer PCR control; 3–6, 100,000 K562 cells untransduced mixed with 0,5,20 and 100 K562 cells known to be positive for the vector MDR-1 sequences; 7–9, negative cells; 10 and 11, bone marrow cells from two breast cancer patients collected after transplantation with CD34⁺ cells exposed to the stromal MDR-1 vector transduction method (positive for MDR-1 vector sequences); 12, peripheral blood cells from one ovarian cancer patient collected after transplantation with CD34⁺ cells exposed to the stromal MDR-1 vector transduction method (positive for MDR-1 vector sequences); 13, positive control; 14, buffer PCR negative control; 15, Phi-X174 molecular weight markers.