Difference between revisions of "Part:BBa K404106"
| (3 intermediate revisions by one other user not shown) | |||
| Line 4: | Line 4: | ||
[[Image:Freiburg10_VectorplasmidBricks 3.png|thumb|center|480px]]<br> | [[Image:Freiburg10_VectorplasmidBricks 3.png|thumb|center|480px]]<br> | ||
| − | + | <h3>Usage and Biology</h3> | |
| − | {| style="margin: 0px 0px | + | {| style="margin: 0px 0px 350px 20px; color: black; float: right;" cellpadding="6" cellspacing="1" border="2" |
! colspan="2" style="background:#66bbff;"|[https://parts.igem.org/Part:BBa_K404106 phTERT promoter] | ! colspan="2" style="background:#66bbff;"|[https://parts.igem.org/Part:BBa_K404106 phTERT promoter] | ||
|- | |- | ||
| Line 30: | Line 30: | ||
<br /> | <br /> | ||
Providing the tumorspecific promoter phTERT with the “Virus Vonstruction Kit”, the iGEM Freiburg_Bioware team 2010 ensures a layer of specificity and safety to the recombinant viral vector system.<br /> | Providing the tumorspecific promoter phTERT with the “Virus Vonstruction Kit”, the iGEM Freiburg_Bioware team 2010 ensures a layer of specificity and safety to the recombinant viral vector system.<br /> | ||
| − | Telomerase activation is a critical step in human tumorigenesis. About | + | Telomerase activation is a critical step in human tumorigenesis. About 90% of human tumors show telomerase activity, while healthy tissue is telomerase negative which means that the protein subunit hTERT is absent in these cells (Cech, 2000), but can be reactivated in cancer cells.<br /> |
Human telomerase reverse transcriptase is driven by the phTERT promoter. Several factors were identified to regulate the hTERT promoter positively and negatively (Kyo et al. 2008). The TATA-less promoter is characterized by enriched GC regions at the putative transcription start region. These are binding sites for the zinc finger transcription factor Sp1, which are clustered upstream of the startcodon (Wick et al. 1999). Within the core promoter (approximately 260 bases upstream of the startcodon) further transcription factor binding sites can be found: The so-called E-boxes (CACGTG) bind the basic helix-loop-helix zipper (bHLHZ) encoded by the Myc familiy (Kyo et al. 2008). Thy c-myc oncogenes are activated by the MAP-kinase pathway induced by EGF-R binding ligands (Maida et al. 2002). A critical factor for regulating hTERT expression is the Activating Enhancer-binding Protein 2 (AP-2) which was found to be an activator of cancer-specific gene expression. Activation of the hTERT promoter was increased by hypoxia described in Nishi et al., 2004 by binding of the HIF-1 alpha transcription factor to the HRE binding sites, still activation cannot be verified in every cancer type. AP-1 acts as a repressor on transcriptional gene expression in humans demonstrated in Takakura, Kyo, Inoue, Wright, & Shay, 2005. | Human telomerase reverse transcriptase is driven by the phTERT promoter. Several factors were identified to regulate the hTERT promoter positively and negatively (Kyo et al. 2008). The TATA-less promoter is characterized by enriched GC regions at the putative transcription start region. These are binding sites for the zinc finger transcription factor Sp1, which are clustered upstream of the startcodon (Wick et al. 1999). Within the core promoter (approximately 260 bases upstream of the startcodon) further transcription factor binding sites can be found: The so-called E-boxes (CACGTG) bind the basic helix-loop-helix zipper (bHLHZ) encoded by the Myc familiy (Kyo et al. 2008). Thy c-myc oncogenes are activated by the MAP-kinase pathway induced by EGF-R binding ligands (Maida et al. 2002). A critical factor for regulating hTERT expression is the Activating Enhancer-binding Protein 2 (AP-2) which was found to be an activator of cancer-specific gene expression. Activation of the hTERT promoter was increased by hypoxia described in Nishi et al., 2004 by binding of the HIF-1 alpha transcription factor to the HRE binding sites, still activation cannot be verified in every cancer type. AP-1 acts as a repressor on transcriptional gene expression in humans demonstrated in Takakura, Kyo, Inoue, Wright, & Shay, 2005. | ||
<br /> | <br /> | ||
| Line 36: | Line 36: | ||
height="auto"/> | height="auto"/> | ||
<br /> | <br /> | ||
| − | |||
| − | |||
</p> | </p> | ||
</html> | </html> | ||
| − | |||
<br /> | <br /> | ||
| − | |||
| − | |||
| − | < | + | <h3>Characterization</h3> |
| + | <p style="margin-right:100px" align="justify"> | ||
| + | For analysis of the submitted BioBrick part, the phTERT promoter was tested for functional biological activity in different cell lines driving gene expression of several genes of interest. | ||
| + | The idea of the iGEM team Freiburg_Bioware 2010 was to produce viral particles encapsidating the transgene expression cassette flanked by the inverted terminal repeats (ITRs). The left ITR is followed by the tumor-specific promoter phTERT, which regulates the gene expression of mVenus, mCherry and the prodrug convertase thymidine kinase fused to the guanylatekinase. Three different human cell lines, AAV-293, HT1080 and A431, were transduced with the viral vectors containing the genes coding for different proteins and promoter activity was analysed by flow cytometry, quantitative real-time PCR and cytotocicity assays (MTT assay) depending on the gene of interest. | ||
| + | </p> | ||
<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K404106 SequenceAndFeatures</partinfo> | <partinfo>BBa_K404106 SequenceAndFeatures</partinfo> | ||
Latest revision as of 14:23, 1 November 2010
phTERT promoter
Usage and Biology
| phTERT promoter | |
|---|---|
| |
| BioBrick Nr. | K404106 |
| RFC standard | RFC 10 |
| Requirement | pSB1C3 |
| Source | synthetic |
| Submitted by | [http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010] |
(telos, end; mere, part)
Providing the tumorspecific promoter phTERT with the “Virus Vonstruction Kit”, the iGEM Freiburg_Bioware team 2010 ensures a layer of specificity and safety to the recombinant viral vector system.
Telomerase activation is a critical step in human tumorigenesis. About 90% of human tumors show telomerase activity, while healthy tissue is telomerase negative which means that the protein subunit hTERT is absent in these cells (Cech, 2000), but can be reactivated in cancer cells.
Human telomerase reverse transcriptase is driven by the phTERT promoter. Several factors were identified to regulate the hTERT promoter positively and negatively (Kyo et al. 2008). The TATA-less promoter is characterized by enriched GC regions at the putative transcription start region. These are binding sites for the zinc finger transcription factor Sp1, which are clustered upstream of the startcodon (Wick et al. 1999). Within the core promoter (approximately 260 bases upstream of the startcodon) further transcription factor binding sites can be found: The so-called E-boxes (CACGTG) bind the basic helix-loop-helix zipper (bHLHZ) encoded by the Myc familiy (Kyo et al. 2008). Thy c-myc oncogenes are activated by the MAP-kinase pathway induced by EGF-R binding ligands (Maida et al. 2002). A critical factor for regulating hTERT expression is the Activating Enhancer-binding Protein 2 (AP-2) which was found to be an activator of cancer-specific gene expression. Activation of the hTERT promoter was increased by hypoxia described in Nishi et al., 2004 by binding of the HIF-1 alpha transcription factor to the HRE binding sites, still activation cannot be verified in every cancer type. AP-1 acts as a repressor on transcriptional gene expression in humans demonstrated in Takakura, Kyo, Inoue, Wright, & Shay, 2005.
Characterization
For analysis of the submitted BioBrick part, the phTERT promoter was tested for functional biological activity in different cell lines driving gene expression of several genes of interest. The idea of the iGEM team Freiburg_Bioware 2010 was to produce viral particles encapsidating the transgene expression cassette flanked by the inverted terminal repeats (ITRs). The left ITR is followed by the tumor-specific promoter phTERT, which regulates the gene expression of mVenus, mCherry and the prodrug convertase thymidine kinase fused to the guanylatekinase. Three different human cell lines, AAV-293, HT1080 and A431, were transduced with the viral vectors containing the genes coding for different proteins and promoter activity was analysed by flow cytometry, quantitative real-time PCR and cytotocicity assays (MTT assay) depending on the gene of interest.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
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.
Maida, Y. et al., 2002. Direct activation of telomerase by EGF through Ets-mediated transactivation of TERT via MAP kinase signaling pathway. Oncogene, 21(26), 4071-9. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12037663.
Nishi, H. et al., 2004. Hypoxia-inducible factor 1 mediates upregulation of telomerase (hTERT). Molecular and cellular biology, 24(13), 6076-83.
Takakura, M. et al., 2005. Function of AP-1 in Transcription of the Telomerase Reverse Transcriptase Gene ( TERT ) in Human and Mouse Cells. Society, 25(18), 8037-8043.