Part:BBa_K404122

[AAV2]-left-ITR_pCMV_betaglobin_mGMK_TK30_hGH_[AAV2]-right-ITR

ITRs

The inverted terminal repeat structures can be subdivided into several palindromic motives: A and A’ form a stem loop which encases B and B’ as well as C and C’. Those motives form both arms of the T-shaped structure. The functional motives on the ITR are two regions that bind Rep 68/78, called Rep-binding elements (RBE on the stem and RBE’ on the B arm) and the terminal resolution site (trs) in which the rep proteins introduce single-stranded nicks. The 3’ OH end of the A motive acts as a primer for DNA replication (Im & Muzyczka, 1990) (Lusby, Fife, & Berns, 1980).

Beta-globin-intron

The beta-globin intron BioBrick consists of a partial chimeric CMV promoter followed by the intron II of the beta-globin gene. The 3´end of the intron is fused to the first 25 bases of human beta globin gene exon 3. The beta globin intron BioBrick is assumed to enhance eukaryotic gene expression (Nott, Meislin, & Moore, 2003). As shown in Figure 9 and Figure 10 the vectorplasmid missing the beta-globin intron showed a negligible difference in mVenus expression compared to viral genomes containing the beta-globin intron. Considering these results and taking into account that a constant volume of viral particles has been used for transduction, the difference between the construct containing and lacking the beta-globin intron is minimal. Since packaging efficiency of the AAV-2 decreases with increasing sizes of the insert (Dong, Fan, & Frizzell, 1996), the iGEM team Freiburg_Bioware suggests using the beta-globin intron in dependence on the size of your transgene.

mGMK_TK30 fusion protein

The thymidine kinase mutant TK30 contains six modified amino acids (Black, Newcomb, Wilson, & Loeb, 1996) created in a first screening showing enhanced affinity for gancivlocir and acyclovir, but reduced specificity for its natural substrate thymidine.

As efficient tumor killing and therefore ganciclovir activation is essential for successful tumor ablation, further improvements were conducted. Overexpression of transgenic thymidine kinase leads to accumulation of non-toxic intermediates, which cannot be phosphorylated sufficiently by endogenous guanylate kinase, the second enzyme in the salvage pathway of nucleotides.
Overcoming this bottleneck was accomplished by fusing the mouse guanylate kinase (mGMK) to the N-terminus of TK30 mutant creating a fusion protein (mGMK_TK30) with enhanced GCV/ACV sensitivity in vitro and in vivo (Ardiani, Sanchez-Bonilla, & Black, 2010) and improved bystander activity. The effect of non-transfected tumor cell killing upon transfer of toxic metabolites through gap junctional intercellular communication (GJIC) or immune-mediated tumor ablation is essential in suicide gene therapy (Pope, 1997). GCV-triphosphate is mainly transported through the central pore formed between connexin proteins from neighboring cells (Gentry, Im, Boucher, Ruch, & Shewach, 2005), but immune-induced bystander effect seems to be likely as well (Grignet-Debrus, Cool, Baudson, Velu, & Calberg-Bacq, 2000).

hGH

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 1319
  Illegal NgoMIV site found at 1977
  Illegal NgoMIV site found at 2882
  Illegal AgeI site found at 3011
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 3413
  Illegal SapI.rc site found at 1369