A novel adenoviral vector carrying an all-in-one Tet-On system with an autoregulatory loop for tight, inducible transgene expresion

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Contents

Introduction

In this research article scientist introduce new Adenoviral vector with Tet-On loop regulation system. Research was made according the adenoviral “All-in-one” Tet-On system issues to regulate gene expression. Although adenoviral vector is one of the most usable vectors in gene therapy, it was hardly compatible to the Tet-On system. There were used new promoters genes and proteins for better reconciliation. Primal idea for this research was suggested as a better way to coordinate gene expression since it would be more convenient.

Methods and technologies

There were created four different new adenoviral Tet-On vectors with different promoters, length of the gene, silencers. Three from four construct have cytomegalavyrus promoter sequence, two of four have double frame reading points in the other words two promoters, all target genes will be poliadenylated in 3’ end. One of four has KRAB expression cassette comparible with Tet genes. All of these vectors were made by using PCR and restriction methods. E1 region was left for target genes.

There were used HEK 293 a human embryonic kidney cells, SW480 a human colon carcinoma cells and U87 MG a human glioblastoma cell line. Moreover they used CHO-K1 a Chinese hamster ovary cell line.

Dual system of Luciferase was used to normalize the cell number and transfection efficiency. After 48 hours of transfection, cells were collected for the test by using dual-luciferase kits provided by Promega. The normalized Luciferase activity was obtained by using the formula: Normalized Luciferase value equal Fly luciferase/ Renilla ludiferase value.

Adenovirus was produced in the HEK293 cell line. Adenovirus “all-in-on” plasmid was made by transfection as a back bone using adenovirus plasmid and linearized constructed sequence. After ten days viral lysates were harvest and propagated in HEK293 and were purified using cesium chloride. The virus particle titers were detected by spectrophotometry at an absorbance at 260nm.

For counting the statistics were used SPSS program packet. Groups were analyzed by one way analysis of variance between groups (ANOVA/LSD). With p value less than 0,05 for significant difference.

Results

The major problem with an adenoviral vector carrying a Tet-On system is the suboptimal regulation of transgene expression. For this reason two newly constructed vectors have a new version of code of the Dox sensitive transactivator.

A novel system of “All-in-one” was evaluated by eGFP in comparing with control. Control in the absence of Dox gene expression was suppressed as well as in the presence of 2µl/ml of Dox. The two most important criteria for evaluating a regulation system are the regulation factor and the maximum induced transgene expression level. To measure these two criteria luciferase gene was cloned into each plasmid. During the experiments it was seen that in the absence of Dox new system is suppressing the genes in the presence of the Dox visible reaction was seen. Measuring the luciferase activity showed significant difference between two phases. Moreover, maximum gene expression level was reach after forty hours of induction of 2µl/ml Dox.

To investigate whether the novel Tet-On system could be able to regulate medical related genes approaches. Into the E1 region of plasmid was inserted TRAIL gene. TRAIL is anticancer agent that selectively induces apoptosis in variety of cancer cells although the TRAIL is toxic for normal cells. For this reason there is some need to strictly regulate the gene expression of the TRAIL in the targeted cell.

Two novel “all-in-one” systems were presented to the host cells, one of the virus show low level of infecting and low cell death ratio due to TRAIL system inside. Another virus has greater infecting ratio and still low level of cell death, both virus results was in the absence of the Dox. In presence of a Dox cell death ratio increased. Although one of the virus loop have revealed greater suppressor activity in the presence of the Dox.

Conclusion

In this study was successfully constructed functional tetracycline inductive adenoviral vector system. Further researches will allow searching for the best properties of “all-in-one” system for better results; moreover this is the first adenovirus-based, all-in-one Tet-on system with large packing size up to 2kb.

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