Wu T, Bloom M L, Yu J M, Tisdale J F, Dunbar C E
Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Hum Gene Ther. 1998 May 20;9(8):1157-64. doi: 10.1089/hum.1998.9.8-1157.
The neomycin phosphotransferase (neo) gene is one of the most common marker genes used in gene transfer experimentation, but potential effects of neo gene expression in vivo have not been systematically investigated. Several early clinical retroviral gene transfer studies have suggested that neo gene expression could have deleterious effects on hematopoiesis, owing to a discrepancy between the level of neo-marked transduced marrow progenitor cells compared with mature circulating progeny cells posttransplantation (Brenner et al., 1993; Kohn et al., 1995; Brenner, 1996b). We examined the long-term in vivo repopulating ability of bone marrow from transgenic mice expressing neo from a strong constitutive promoter using a competitive repopulation assay. Different ratios of neo transgenic and wild-type congenic marrow cells were cotransplanted into W/Wv recipient mice. The percentages of blood cells containing the neo transgene in each group of recipient mice monitored for 4 months posttransplantation closely matched the input ratios of neo transgenic to congenic control marrow cells. Similar concordances of engraftment with input ratios of neo transgenic cells were also found in spleen, thymus, and whole marrow of recipient mice at 4 months posttransplantation. Analysis of the beta-hemoglobin phenotype (beta(single) for the neo transgenic and C57 control cells and beta(diffuse) for the congenic competitor HW80 cells) in recipients confirmed erythroid 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 results suggest that the low-level contribution of vector-marked cells to circulating populations in clinical trials is not due to direct deleterious effects of neo gene expression on hematopoiesis.
新霉素磷酸转移酶（neo）基因是基因转移实验中最常用的标记基因之一，但neo基因在体内表达的潜在影响尚未得到系统研究。一些早期的临床逆转录病毒基因转移研究表明，neo基因表达可能对造血功能产生有害影响，这是由于移植后neo标记的转导骨髓祖细胞水平与成熟循环子代细胞水平之间存在差异（Brenner等人，1993年；Kohn等人，1995年；Brenner，1996b）。我们使用竞争性再增殖试验检测了来自表达neo的转基因小鼠的骨髓在体内的长期再增殖能力，该neo由一个强组成型启动子表达。将不同比例的neo转基因和野生型同基因骨髓细胞共移植到W/Wv受体小鼠中。在移植后4个月监测每组受体小鼠中含有neo转基因的血细胞百分比，其与neo转基因与同基因对照骨髓细胞的输入比例密切匹配。在移植后4个月，在受体小鼠的脾脏、胸腺和全骨髓中也发现了neo转基因细胞的植入与输入比例的类似一致性。对受体中β-血红蛋白表型（neo转基因细胞和C57对照细胞为β（单一），同基因竞争细胞HW80细胞为β（弥散））的分析证实，neo转基因骨髓细胞的红系再增殖水平与输入比例相匹配。我们得出结论，表达neo的造血细胞在体内没有可检测到的植入或成熟缺陷。这些结果表明，在临床试验中，载体标记细胞对循环群体的低水平贡献并非由于neo基因表达对造血功能的直接有害影响。
