Difference between revisions of "Part:BBa K782063"

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CMV promoter_mouse guanylate kinase_thymidine kinase 30

Introduction

Figure 2: Assembly of mGMK_TK30 (BBa_K404113) under the control of CMV promoter. Insert (mGMK_TK30, (BBa_K404113)) was digested with EcoRI and SpeI and pcDNA3 vector with EcoRI and XbaI, loaded on an agarose gel and the appropriate bands were purified. Vector and insert were ligated and transformed into E. coli strain DH5α. The DNA sequence of the resulting plasmid pPCMV-mGMK_TK30 was confirmed by DNA sequencing.

Suicide gene therapy or gene-directed enzyme prodrug therapy (GDEPT) is widely used in cancer treatment. One of the most studied GDEPT systems is the herpes simplex virus thymidine kinase (HSV-TK) with purine nucleoside analog ganciclovir (GCV) as a prodrug. Systemic administration of the prodrug ganciclovir induces apoptosis only in cells transfected with HSV-thymidine kinase while the untransfected cells survive. Unlike human thymidine kinase, HSV-thymidine kinase is able to phosphorylate ganciclovir to form ganciclovir-monophosphate, which is then phosphorylated to ganciclovir-diphosphate and then to ganciclovir-triphosphate. Ganciclovir-triphosphate is then incorporated into DNA, which causes inhibition of DNA synthesis and subsequently leads to apoptosis (Ardiani et al., 2010). Following the BioBrick Standard Assembly technique iGEM team Slovenia inserted the mouse guanylate kinase – thymidine kinase fusion gene (mGMK_TK30, (BBa_K404113)) under the control of CMV promoter. HSV-TK mutant (TK30) contains six amino acid substitutions and shows enhanced sensitivity to ganciclovir (Kokoris et al., 1999), while mouse guanylate kinase improves phosphorylation of ganciclovir-monophosphate to ganciclovir-diphosphate, thus preventing accumulation of ineffective intermediate products (i.e. GCV-MP, GCV-DP) due to limited ability of endogenous guanylate kinase (Ardiani et al., 2010).We transfected HEK293 cells with the pPCMV-mGMK_TK30 plasmid and monitored cell survival after the addition of gancyclovir. We observed cells under an optical microscope daily and were attentive to any morphological signs of decreased viability. Additionally we stained cells with Hoechst and SytoxGreen514 dye to distinguish between live and dead cells with the help of a confocal microscope. We quantified viable cell numbers at different time points by cell counting and flow cytometry.

Figure 2: Mechanism of action of the HSV-TK/GCV system.

Characterization

We tested different amounts of transfected pPCMV-mGMK_TK30 DNA, different concentrations of ganciclovir and different time periods after addition of ganciclovir to find the optimal conditions where our pCMV-mGMK_TK30-transfected cells would be most efficiently killed while untransfected cells would be left unharmed. We monitored pPCMV-mGMK_TK30-transfected ganciclovir-treated cells under a light microscope and discovered that as little as 40 hours of incubation with higher concentrations of ganciclovir (>30 μg/ml) is enough to kill over 50 % of our pPCMV-mGMK_TK30-transfected cells. After 65 hours of incubation with ganciclovir there was a decrease in cell number and survival already at low concentration of ganciclovir (1 μg/ml).

Interestingly we also observed that higher amounts of transfected mGMK_TK30 influences cell viability even without ganciclovir. Also higher concentrations of ganciclovir became cytotoxic after longer periods of incubation. But more importantly, we showed that the HSV-TK/GCV system successfully kills cells even at low amounts of transfected mGMK_TK30 and upon addition of relatively low concentrations of ganciclovir (5 μg/ml).

We determined the number of viable cells after ganciclovir treatment by staining the cells with Trypan Blue (to distinguish live cells from the dead) and counting them. After 6 days of incubation with ganciclovir, the cytotoxic effect could be seen when only 1 μg/ml ganciclovir was added to pPCMV-mGMK_TK30 transfected cells while untransfected cells stayed unharmed. The number of viable cells decreased to <10 % when 10 μg/ml ganciclovir was added. These results demonstrate that our safety mechanism is effective even at low doses of ganciclovir, when only therapeutic cells are killed while untransfected cells are left unharmed. Sequence and Features