Comparison of the Expression of a Mutant Dihydrofolate Reductase under Control of Different Internal Promoters in Retroviral Vectors

Abstract

To determine the effect of different promoters on the expression of an altered dihydrofolate reductase (DHFR) gene conferring methotrexate (MTX) resistance in different cell types, double-copy retroviral vectors were constructed carrying a murine mutant DHFR under the control of five different promoters, i.e., human adenosine deaminase (ADA), simian virus 40 (SV40), thymidine kinase (TK), human beta-actin, and cytomegalovirus (CMV). Their expression was compared in NIH-3T3 cells, three human leukemia cell lines, and mouse bone marrow. The variant DHFR is readily expressed from these various promoters in retroviral vectors at a selectable level. In 3T3 cells, the DHFR constructs containing the SV40 promoter conferred the highest levels of resistance to MTX. In K562 and Raji cells, the construct with the TK promoter produced the highest level of resistance. However granulocyte-macrophage colony-forming unit (CFU-GM) colonies from mouse marrow were more resistant to MTX when infected with vectors containing the SV40 promoter and ADA promoter as compared to the other promoter constructs. These studies show that mouse fibroblast cell lines such as NIH-3T3 do not predict the effectiveness of retroviral-mediated gene transfer for marrow progenitor cells, and that the activity of retroviral vector-encoded promoters vary in an unpredictable manner from cell type to cell type. Possible implications for basic gene transfer studies and clinical applications are discussed.

Probing the Folate-binding Site of Human Thymidylate Synthase by Site-directed Mutagenesis

“…(colony-forming unitgranulocyte macrophage) and HPP-CFC (high proliferative potentialcolony forming cell) assays were carried out as described previously (35,36). Briefly, bone marrow cells from CBA/J (7 to 11 weeks of age, male) mice were harvested in Isocove's modified Dulbecco's medium and 20% fetal calf serum.…”

Retroviral vector design studies toward hematopoietic stem cell gene therapy for mucopolysaccharidosis type I

“…13 To enhance and stabilize the expression of transgenes in hematopoietic cells, some investigators have inserted into vector constructs promoters of certain housekeeping genes such as ␤-actin, 14,15 thymidine kinase, 16 and phosphoglycerate kinase (PGK). 17,18 However, data on parallel comparisons and evaluations of promoter strength have been insufficient due to differences in the reporter genes and retroviral vectors employed, and limited information on transduction frequency.…”

Co-expression of the herpes simplex virus thymidine kinase gene potentiates methotrexate resistance conferred by transfer of a mutated dihydrofolate reductase gene

“…After the 3-day selecin the 5′-LTR, to form a double copy structure. 25,26 This tion, cells were plated in 96-well plates, and cytotoxicity vector was digested with BclI at 65°C overnight, bluntmeasured by a colorimetric assay using XTT, sodium 3′-ended with Klenow enzyme, and then cut with XhoI. A (1-((phenylamino)-carbonyl)-3,4-tetrazolium)-bis(4-methHSVtk-containing vector, TEL-1 (kind gift of Dr Michel oxy-6-nitro)benzene-sulfonic acid hydrate, as described Sadelain, MSKCC) 27 was digested with XbaI/XhoI, and previously.…”