Transient expression of dPH/R364E/dC facilitated HSC/HPC engraftment under myeloablative or nonmyeloablative conditions. (A) Enhanced engraftment under myeloablative conditions demonstrated by a competitive repopulation assay. Ly5.1⁺ cells were transfected with control or dPH/R364E/dC-expressing vector by electroporation and injected into lethally irradiated Ly5.2⁺ mice together with Ly5.2⁺ competitor cells. Peripheral-blood cells of recipient mice that underwent transplantation with cells (○ and •, 0.6 × 10⁶; ▵ and ▴, 2.4 × 10⁶ cells) transfected with control vector (○ and ▵) or with dPH/R364E/dC-expressing vector (• and ▴) were stained and analyzed at indicated time points. Results of 2 experiments are shown. (B) Early platelet production from progenitors transiently expressing dPH/R364E/dC mutant. BM cells from GFP-transgenic mice were transfected and injected into lethally irradiated mice. GFP-positive platelet counts in animals that received cells transfected with control vector (□) or dPH/R364E/dC expression vector (▪) were measured 10 or 21 days after transfer. Shown are means ± SD of results obtained from 2 transfer experiments. ^*P < .01. (C) Enhanced engraftment under nonmyeloablative conditions and reconstitution of the lymphoid compartment of SCID mice. Transfected wild-type cells were washed and intravenously injected into SCID mice treated with 1.0 Gy nonmyeloablative irradiation. Peripheral-blood cells obtained from untreated SCID mice (top left) or from mice that received transplants with cells treated with control vector (top middle) or dPH/R364E/dC-expressing vector (top right) were stained and analyzed 8 weeks after transplantation. The percentage of B220⁺ B-lineage or CD3⁺ T-lineage cells derived from donor cells transfected with control vector (bottom panels, ○) or with dPH/R364E/dC plasmid (•), harvested at the indicated time points, was compared with that of normal wild-type cells (▪). Results obtained from 2 transfer experiments are shown. ^*P < .03. (D) Lack of plasmid DNA integration into genomic DNA of reconstituted lymphocytes. Genomic DNA isolated from peripheral-blood nucleated cells of recipients at the indicated time points was subjected to PCR amplification for the neomycin-resistant gene derived from the plasmid DNA or for the Rag2 gene for control. V indicates mice that received transplants with vector-transfected cells; DN, mice that received transplants with dPH/R364E/dC-transfected cells. Representative results of multiple experiments are shown.