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The use of an engineered thymidylate synthase gene for gene therapy applications
Author(s) -
Morrey Erin L,
Spencer H. Trent
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.lb474
Subject(s) - transduction (biophysics) , selectable marker , haematopoiesis , biology , genetic enhancement , transfection , population , stem cell , progenitor cell , microbiology and biotechnology , cell culture , gene , genetics , transgene , biochemistry , medicine , environmental health
The effectiveness of many gene therapy applications is limited by the efficiency of genetic modification of target cells. For example, clinical trials have shown that the transduction efficiency of hematopoietic stem cells using recombinant retroviruses is extremely low compared to the efficiency of transducing hematopoietic progenitor cells. One method for increasing the percentage of genetically‐modified cells is to selectively expand the population after transduction. We examined the potential for using an engineered thymidylate synthase construct, designed to be optimally expressed in mammalian cells (OPTecTS), as a drug‐resistance marker to preferentially select genetically‐modified cells. An MSCV‐OPTecTS construct was developed and shown to confer extremely high‐level antifolate resistance to transduced cells using several in vitro assays. OPTecTS transduced cells were enriched to a high percentage of the total cell population after only five days in culture. Also, co‐transfection of OPTecTS with GFP resulted in the selection of GFP+ cells after selection, and PCR confirmed the presence of the OPTecTS DNA in all cases, indicating that the OPTecTS construct was directly protecting the GFP+ cells. These results indicate that OPTecTS can be used for the in vitro selection of modified cells, not only as a selectable marker for gene therapy but also for molecular biology applications.