James R I, May C, Vagt M D, Studebaker R, McIvor R S
Institute of Human Genetics, and Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis 55455, USA.
Exp Hematol. 1997 Nov;25(12):1286-95.
Expression of the arg22, drug-resistant variant of dihydrofolate reductase (DHFR) in hematopoietic cells has been demonstrated to confer resistance to methotrexate (MTX) in mice, even though this variant suffers from low catalytic activity. The recently reported tyr22 variant has the advantage of higher catalytic activity combined with significant resistance to MTX. To evaluate the resistance conferred by tyr22-DHFR in vivo, we generated several transgenic mouse lines carrying a tyr22-DHFR minigene regulated by its natural promoter. The transgene copy number in 11 lines ranged from 6 to 68 copies and ribonuclease protection analysis demonstrated that 4 of these lines expressed significant transgenic DHFR mRNA at 20 to 68% of the endogenous DHFR mRNA level. Marrow from 4 of the 11 lines conferred significant increases in MTX-resistance in comparison with normal marrow when transplanted into lethally irradiated recipients. The ability of the tyr22-DHFR transgenic marrow to confer MTX-resistance to bone marrow transplant (BMT) recipients did not correlate with the level of mRNA expression or the number of transgene copies. However, two lines (lines 11 and 15) that were most effective in maintaining normal hematocrit levels in BMT recipients receiving 1 mg/kg/day MTX exhibited the greatest ability to form MTX-resistant hematopoietic progenitor colonies in vitro. Furthermore, MTX dose escalation studies demonstrated that line 11 marrow conferred resistance in BMT recipients receiving up to 6 mg/kg/day MTX. Southern blot analysis of the BMT recipients 7 months posttransplantation showed a preponderance of transgenic donor-derived cells in bone marrow and spleen, as well as a surprisingly high level in the small intestine. These results indicate that tyr22-DHFR is likely to be superior to arg22-DHFR in conferring MTX-resistance in BMT recipients, illustrating its usefulness for chemoprotection during MTX chemotherapy and also potentially for in vivo selection of transduced cells in gene therapy trials.
已证实，即使二氢叶酸还原酶（DHFR）的精氨酸22耐药变体催化活性较低，但在造血细胞中表达时仍能使小鼠对甲氨蝶呤（MTX）产生耐药性。最近报道的酪氨酸22变体具有较高催化活性以及对MTX显著耐药的优势。为了评估酪氨酸22 - DHFR在体内赋予的耐药性，我们构建了几个携带由其天然启动子调控的酪氨酸22 - DHFR小基因的转基因小鼠品系。11个品系中的转基因拷贝数在6至68个拷贝之间，核糖核酸酶保护分析表明，其中4个品系表达的转基因DHFR mRNA水平显著，为内源性DHFR mRNA水平的20%至68%。当将11个品系中的4个品系的骨髓移植到接受致死性照射的受体中时，与正常骨髓相比，其赋予的MTX耐药性显著增加。酪氨酸22 - DHFR转基因骨髓赋予骨髓移植（BMT）受体MTX耐药性的能力与mRNA表达水平或转基因拷贝数无关。然而，在接受1 mg/kg/天MTX的BMT受体中，最能有效维持正常血细胞比容水平的两个品系（11号和15号品系）在体外形成MTX耐药造血祖细胞集落的能力最强。此外，MTX剂量递增研究表明，11号品系的骨髓能使接受高达6 mg/kg/天MTX的BMT受体产生耐药性。对移植后7个月的BMT受体进行Southern印迹分析显示，骨髓和脾脏中主要是转基因供体来源的细胞，小肠中的水平也出奇地高。这些结果表明，在赋予BMT受体MTX耐药性方面，酪氨酸22 - DHFR可能优于精氨酸22 - DHFR，这说明它在MTX化疗期间的化学保护以及基因治疗试验中体内转导细胞的选择方面可能有用。
