Part:BBa_K404126
[AAV2]-left-ITR_pCMV_betaglobin_CD_hGH_[AAV2]-right-ITR
| [AAV2-left-ITR_pCMV_betaglobin_CD_hGH_[AAV2]-right-ITR ] | |
|---|---|
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| BioBrick Nr. | BBa_K404126 |
| RFC standard | RFC 10 |
| Requirement | pSB1C3 |
| Source | pAAV_MCS: provided by Stratagene |
| Submitted by | [http://2010.igem.org/Team:Freiburg_Bioware 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 gene of interest (GOI)
cytosine
deaminase ) was
inserted into the transgene expression cassette and its biological
activity was
test in cell culture. In E. coli, Cytosine
Deaminase (CD) (EC
3.5.4.1) is encoded by codA. The protein plays a crucial role in
nucleotide
synthesis since it catalyzes the deamination of cytosine to uracil
(Danielsen et al. 1992).
In eukaryotic cells, expression of this protein can be used to convert
the
non-toxic prodrug 5-fluorocytosine to the toxic compound
5-fluorouracil. The
presence of this nucleotide analogue in the target cell blocks the
synthesis of
thymidine by inhibition of the essential enzyme thymidylate synthase,
therefore
leading to cell death.
Furthermore, another advantage of 5-FUra is its solubility and ability to freely diffuse into adjacent cells [Domin et al., 1993; Huber et al., 1994] therefore enhancing the bystander effect.
References:
Danielsen, S. et al., 1992. Characterization of the Escherichia coli codBA operon encoding cytosine permease and cytosine deaminase. Molecular microbiology, 6(10), pp.1335-44. Available at: http://www.ncbi.nlm.nih.gov/pubmed/1640834.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1319
Illegal AgeI site found at 2606 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 3008
//viral_vectors/aav
//viral_vectors/aav/vector_plasmid
| None |