The activation of certain gene promoters to drive transgene expression after proton irradiation allows the development of methods for noninvasive, high-resolution targeting of CNS tumors. These gene promoters can be designed for treatment and visualization of treatment therapies. Herpes simplex virus type 1 thymidine kinase (HSV1-tk) is both a marker gene used frequently for molecular imaging in positron emission tomography (PET) and a cytotoxic gene converting ganciclovir (GCV) to induce apoptosis in the targeted cellular population. It has been shown that a mutant version called HSV1-sr39tk is a better marker due to its higher enzymatic activity. Therefore we set out to create a HSV1-sr39tk marker using site-directed mutagenesis and transfect murine C6 glioma cell lines with both markers for in vitro analysis. The cloning vector pCR-Blunt II-TOPO (Invitrogen) was used and transformed into DH5a E. coli (Invitrogen). Plasmids were extracted via miniprep protocol (Qiagen) and assessed by restriction enzyme digests using EcoR1 and BglII (Fisher Scientific) on a 1% agarose gel (SeaKem LE). DNA concentration was also quantified with UV-spectrophotometry. The PCR method of site-directed mutagenesis was performed using primers ordered from IDT Inc. and followed protocols described in Quikchange II Site-Directed Mutagenesis Kit (Stratagen). End products were transformed, extracted, and quantified for sequence analysis (Davis Sequencing) prior to transfection into C6 glioma cells. The HSV1-tk strain was also sequenced and transfected into the murine cells. Both cell lines were then selected using Zeocin (Invitrogen). In vitro testing included sensitivity of cell lines to GCV to construct growth inhibition curves and analysis of the bystander effect in cells treated with GCV. Preliminary molecular and cellular data suggest that the gene promoter is now ready for in vivo tests, using rodents as the next step toward building a model for visualization of proton targeting in cancer therapy.
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