Part:BBa_K404124

[AAV2]-left-ITR_phTERT_betaglobin_mGMK_TK30_hGH_[AAV2]-right-ITR

Recombinant Viral Particles for the Virus Construction Kit

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. 

After assembly of plasmids containing all required elements, vector plasmid functionality was confirmed in cell culture. AAV-293 cells stably expressing the E1A and E1B proteins were transfected with three plasmids (pHelper, pRC, pGOI). Virus particles were harvested 72 hours post transfection and the tumor cell line HT1080 was transduced with the recombinant viral vectors encapsidating the gene of interest mVenus (BBa_I757008).

Tumor-specific promoter phTERT

Several assembly steps have to be performed in order to obtain the fully assembled vector plasmid. Facilitating the cloning steps, the iGEM team Freiburg_Bioware 2010 provide the 5´nucleotide components in respect to the desired gene of interest which are the left inverted terminal repeat (BBa_K404100) followed by the phTERT promoter (BBa_K404106). By providing this 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 (Cech 2000). In most somatic cells, the phTERT promoter is inactive (Wick et al. 1999). This prevents expression of the hTERT protein subunit and renders the healthy tissue telomerase negative (Kyo et al. 2008).

 

Reference

Cech, T., 2000. Life at the End of the Chromosome: Telomeres and Telomerase. Angewandte Chemie (International ed. in English), 39(1), 34-43. Available at: http://www.ncbi.nlm.nih.gov/pubmed/10649348.

Kyo, S. et al., 2008. Understanding and exploiting hTERT promoter regulation for diagnosis and treatment of human cancers. Cancer science, 99(8), 1528-38. Available at: http://www.ncbi.nlm.nih.gov/pubmed/18754863.

Wick, M., Zubov, D. & Hagen, G., 1999. Genomic organization and promoter characterization of the gene encoding the human telomerase reverse transcriptase (hTERT). Gene, 232(1), 97-106. Available at: http://www.ncbi.nlm.nih.gov/pubmed/10333526.

 

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1122
  Illegal NgoMIV site found at 1780
  Illegal NgoMIV site found at 2685
  Illegal AgeI site found at 2814
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 3216
  Illegal SapI.rc site found at 1172