MURINE BONE-MARROW EXPRESSING THE NEOMYCIN RESISTANCE GENE HAS NO COMPETITIVE DISADVANTAGE ASSESSED IN-VIVO

The neomycin phosphotransferase (neo) gene is one of the most common m arker genes used in gene transfer experimentation, but potential effec ts of neo gene expression in vivo have not been systematically investi gated. Several early clinical retroviral gene transfer studies have su ggested that neo gene expression could have deleterious effects on hem atopoiesis, owing to a discrepancy between the level of neo-marked tra nsduced marrow progenitor cells compared with mature circulating proge ny cells posttransplantation (Brenner et al., 1993; Kohn et al., 1995; Brenner, 1996b), We examined the long-term in vivo repopulating abili ty of bone marrow from transgenic mice expressing neo from a strong co nstitutive promoter using a competitive repopulation assay, Different ratios of neo transgenic and wild-type congenic marrow cells were cotr ansplanted into W/W-v recipient mice. The percentages of blood cells c ontaining the neo transgene in each group of recipient mice monitored for 4 months posttransplantation closely matched the input ratios of n eo transgenic to congenic control marrow cells. Similar concordances o f engraftment with input ratios of neo transgenic cells were also foun d in spleen, thymus, and whole marrow of recipient mice at 4 months po sttransplantation, Analysis of the beta-hemoglobin phenotype (beta(sin gle) for the neo transgenic and C57 control cells and beta(diffuse) fo r th, congenic competitor HW80 cells) in recipients confirmed erythroi d repopulation from neo transgenic marrow cells at levels matching the input ratios, We conclude that hematopoietic cells expressing neo had no engraftment or maturation defects detectable in vivo. These result s suggest that the low-level contribution of vector-marked cells to ci rculating populations in clinical trials is not due to direct deleteri ous effects of neo gene expression on hematopoiesis.