Transgenic mice expressing the tyr22 variant of murine DHFR: protection of transgenic marrow transplant recipients from lethal doses of methotrexate.
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.
James
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Institute of Human Genetics, and Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis 55455, USA.
May
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Vagt
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MD
Studebaker
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McIvor
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RS
D019943:Amino Acid Substitution; D000818:Animals; D016026:Bone Marrow Transplantation / Q000379:methods*; D003114:Colony-Forming Units Assay; D019008:Drug Resistance, Neoplasm; D005260:Female; D005493:Folic Acid Antagonists / Q000008:administration & dosage*; D006410:Hematopoiesis; D008727:Methotrexate / Q000008:administration & dosage*; D051379:Mice; D008822:Mice, Transgenic; D012333:RNA, Messenger / Q000235:genetics; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics*; D014443:Tyrosine
D005493:Folic Acid Antagonists; D012333:RNA, Messenger; D014443:Tyrosine; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate