Recipient mice then received a lethal irradiation dose of 900 rads to eliminate host bone marrow
Recipient mice then received a lethal irradiation dose of 900 rads to eliminate host bone marrow. in reporter cell lines compared with wild type. Retention of the probe L-FMAU was 18-fold higher in hdCK3mut cells compared with WT hdCK (Fig. 1 and = 0.027, L-FMAU = 0.0052) (side is control L1210-10K (dotted collection). side is usually L1210-10K cells with stable expression of WT dCK or hdCK3mut (solid collection). (= 0.0006). Enzyme kinetic analysis further exhibited high substrate affinity of hdCK3mut to L-FMAU with a measured = 0.0006) compared with WT hdCK grafts (Fig. 1and Fig. S2). These results decided that hdCK3mut and L-FMAU make a suitable PET reporter gene and probe combination for in vivo studies. Expression of hdCK3mut in Mouse HSCs Allow Noninvasive Detection of Reporter Cell Transplantation Before Normalization of Peripheral Blood Counts. A competitive mouse bone marrow transplantation (BMT) study was MK-1775 chosen to test whether hdCK3mut can detect transplanted cells during early hematopoietic reconstitution (24C28). Donor cells were generated by treating mice with 5-flourouracil 5 d preharvest for HSC enrichment. Collected bone marrow was retrovirally infected with 40C60% transduction efficiency to express hdCK3mut (coexpressed with YFP through an IRES) or the control of IRES-YFP only (Fig. S1). Recipient mice then received a lethal irradiation dose of 900 rads to eliminate host bone marrow. Mice were transplanted with the mixed populace of reporter/nonreporter HSC-enriched donor bone marrow (Fig. 2 0.05). Under standard conditions mice will display normalized engraftment and total blood counts (CBC) within 8 wk after BMT (27). We hypothesized that early engraftment and growth could be monitored by reporter imaging before normalization of peripheral blood measurements. At 4 wk post-BMT, animals received PET/CT scans with [18F]-FDG and the following day [18F]-L-FMAU (Fig. 2 and and Fig. S3). Animals in the control YFP cohort experienced no hematopoietic transmission observed with [18F]-L-FMAU (Fig. 2 0.05) higher accumulation of [18F]-L-FMAU compared with unlabeled cells in all hematopoietic tissues (Fig. 2and Figs. S4 and S5). Circulation cytometry analysis evaluated MK-1775 the spleen, thymus, bone marrow, and peripheral blood for total donor engraftment by lineage, reporter expression (YFP expression), and cell cycle. A representative fluorescent-activated cell sorting (FACS) plot of hdCK3mut engraftment within the spleen is usually displayed (Fig. 3and Fig. S8). Total human engraftment was detected with human-specific HLA staining. Sequential sections verified reporter positive cells by anti-dCK and anti-YFP staining. Anti-dCK IHC in the spleen stained a portion of the total engrafted human cells, consistent with the peripheral blood FACS (Fig. 4and em D /em ) hdCK3mut-engrafted animals. Lineage-specific integrations recognized committed progenitor cells. Integration sites found in all three populations are derived from a common transduced cell of origin (HSC) which then differentiated into all lineages (Fig. 5 em B /em C em D /em ). Previous vector copy number per cell was determined by PCR to be 0.5 (0.485, hdCK3mut; 0.494, YFP) after transduction. It is estimated that each HSC Mouse monoclonal to CD95(Biotin) integration site represents a single engrafted clone. A comparison of the total quantity of integrations from each sample in hdCK3mut and YFP animals confirms that the number of integrations detected is similar between vectors. This demonstrates that this expression of hdCK3mut does not prevent hHSCs from differentiating into all major hematopoietic lineages within the humanized mouse model. hdCK3mut also experienced no effect on long-term MK-1775 engraftment and was detected up to 5 mo post-HSC transplant with no lineage restriction due to gene toxicity or clonal growth due to growth advantage. Discussion We have demonstrated that an alternate dCK mutant (hdCK3mut) is usually well-tolerated, highly sensitive, and capable of monitoring long-term HSC engraftment. Expression of HSV1-TK After Gene Transfer Provides a Security Mechanism in Aberrant Reporter Cell Populations Through Reporter-Specific Cytoxicity (38, 39). hdCK3mut provides an alternative PET reporter gene to sr39TK. One.