|RFC standard||RFC 10|
|Source||pAAV_MCS: provided by Stratagene|
|Submitted by||FreiGEM 2010|
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. While mCherry is expressed in the cytosol, mVenus can be detected in nuclei due to the VP1 integrated NLS.
Movie 1:HT1080: mVenus (nuclei) and mCherry (cytosol) expression
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000Illegal BsaI site found at 2580