Difference between revisions of "Part:BBa K404129"

Producing recombinant virus particles for therapeutical applications is, besides specific cell targeting, purification and quantification assays of AAV-2, one intention of the Virus Construction Kit provided by the iGEM team Freiburg_Bioware 2010. For obtaining a modular toolkit, the complex biological system of the Adeno-associated virus serotype 2 was examined by an exhaustive literature search. Subsequently, the essential components for AAV-2 particle production were extracted and redesigned to match the iGEM standard.

The provided tripartite system is independent of a superinfection  of Adeno- or herpes simplex viruses since the genes encoding the required helper-proteins are co-transfected. Inside the eukaryotic host cell, the DNA sequence containing the inverted terminal repeats (ITRs) is extracted and later encapsidated into the preformed capsids after production of single-stranded DNA. Consequently, this plasmid is known as the vector plasmid (pGOI). Promoter, beta-globin intron and the hGH terminator signal are flanked by the ITRs (ITRs, BBa_K404100 and BBa_K404101) and regulate transgene expression. The vector plasmid containing the desired gene of interest is cotransfected with the RepCap plasmid (BBa_K404001, BBa_K404002 or BBa_K404003) and the pHelper plasmid. To obtain the fully assembled vector plasmid, several assembly steps have to be performed. 

The Composite part [AAV2]-left-ITR_pCMV_betaglobin_mCherry_hGH_[AAV2]-right-ITR provided by the iGEM team Freiburg_Bioware comprises all cis-elements required for efficient transgene expression in mammalian cells (polyadenylation terminator: BBa_K404108 and transcription enhancer element: BBa_K404107) and encapsidation into virus particles (ITRs: BBa_K404100 and BBa_K404101). The mCherry coding sequence as gene of interest enables facile detection of transduced cells using flow cytometry or fluorescence microscopy.

Furthermore, by providing the tumor-specific promoter with the “Virus Vonstruction Kit”, the iGEM Freiburg_Bioware team 2010 ensures another layer of specificity and safety to the recombinant viral vector system. Telomerase activation is a critical step in human tumorigenesis and about 90% of human tumors show telomerase activity. In most somatic cells, the phTERT promoter is inactive. This prevents expression of the hTERT protein subunit and renders the healthy tissue telomerase negative.