The last doses of chemotherapy had been given, to allow assessment of the relative survival of the GFP transduced cells nattokinase compared to the NoNtransduced cells. The data for the numbers of CD34 cells that were transduced, the percentages of cells that expressed GFP by flow cytometry before transplantation, the vector copy numbers per cell, the numbers of colonies grown from each CD34 cell fraction, and the percentage of colonies that expressed GFP for the third series of transplants are shown. CD34 cell dosages were between 3.7 and 6.8 × 106 cells, which are within the range typically used in human transplants. GFP expression glycyrrhetin from cells transduced with the SIV GFP vector ranged between a low of 6.2% to a high of 39.3%. The vector copies/cell measured from the CD34 cells immediately after transduction ranged from 1.8 to 204 copies/cell. These high values are questionable and may in part represent nonintegrated reverse transcription products and possibly residual vector plasmids from the packaging process present in or on the CD34 cells shortly after transduction.
Two of the marrow samples grew very few colonies, whereas the other samples produced between 12 to 71 colony forming unit granulocyte macrophage custom peptide synthesis from the plated sample. Between 47 and 57% of the colony forming unit granulocyte macrophage grown from the CD34 cells transduced with the SIV GFP vector expressed the GFP transgene. To further characterize the relationship between vector copy numbers assessed immediately or after a 2 week short term culture of the cells, CD34 cells from a rhesus monkey donor of similar age were transduced with either the SIV NoN and SIV GFP vectors and analyzed for vector copy number 1 2 days after transduction and after a 2 week culture. We found that the vector copy number measured immediately after transduction grossly MG-341 overestimated the gene transfer.. We also plated colony forming units and determined the percentage that were vector provirus positive by PCR and the percentage of cells that expressed GFP by flow cytometry.
75% of the CFU grown from the CD34 cells transduced with the GFP vector contained the vector as determined by PCR and 30% of the cells expressed GFP as determined by flow cytometry. Gene marking of blood and bone marrow cells Following autologous transplants, samples of peripheral blood and bone marrow were obtained monthly. Cell subpopulations were isolated by ficoll gradient separation and immunomagnetic methods. DNA was extracted to measure the levels of cells containing each of the vectors, and assayed by qPCR using primers and probes to the NoN and GFP sequences. At the time of tissue harvest,large volumes of blood were obtained and additional leukocyte subsets were isolated and assayed by qPCR for the levels of gene marking. The gene marking data from the six recipients treated in Group 3 are shown in Figure 5. Recipient 3B displayed the highest and most consistent gene marking in blood and bone marrow cells of any of the treated animals. In this recipient, the levels of GFP marked cells consistently ranged between 0.001 and 0.01 vector copies/cell. Recipient 3D had GFP marking at 0.0001 0.001 vector copies/cell but no NoN marking. Recipients 3E and 3F showed marking with the NoN vector at 0.001 0.01 vector copies.