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| | </header> | | </header> |
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| − | <h3 class="inner-h">Favorite composite part——CMV-AR185-T2A -eGFP-polyA</h3> | + | <h3 class="inner-h">Favorite composite part——Left ITR-CMV-AR185-T2A-EGFP-Poly(A)-Right ITR</h3> |
| | <p class="inner-text"> | | <p class="inner-text"> |
| − | For this year’s iGEM competition, our favorite composite part is C185, short for CMV-AR185-T2A -eGFP-polyA, This part is composed of CMV promotor, AR185 linked with eGFP by T2A and poly A. | + | For this year's iGEM competition, our favorite composite part is Left ITR-CMV-AR185-T2A-EGFP-Poly(A)-Right ITR. |
| | </p> | | </p> |
| | + | <p class="inner-text"> |
| | + | This device is a shuttle plasmid of the AAV helper-free system. It consists of a CMV promoter, nanobody AR185-T2A-EGFP (BBa_K2865001), SV40 polyA signal and flanked by two inverted terminal repeats (BBa_K2865002 and BBa_K2865003). |
| | + | </p> |
| | + | <p class="inner-text"> |
| | + | The use of this part is 1) to produce AAV9 particles, 2) to express our novel intrabody AR185 linked with reporter EGFP in failing heart, and 3) to test its efficacy for heart failure. |
| | + | </p> |
| | + | |
| | <div style="text-align:center" class="resultimage"> | | <div style="text-align:center" class="resultimage"> |
| | <img src="https://static.igem.org/mediawiki/2018/e/e6/T--SMMU-China--Composite_part_Pic_1.png" style="width: 60%;"> | | <img src="https://static.igem.org/mediawiki/2018/e/e6/T--SMMU-China--Composite_part_Pic_1.png" style="width: 60%;"> |
| | <p style="font-style: italic;text-align: center;padding: 0em 100px 1em;"> | | <p style="font-style: italic;text-align: center;padding: 0em 100px 1em;"> |
| − | <strong>Figure 1. The C185 construct</strong> | + | <strong>Figure 1. Schematic diagram of constructs of Left ITR-BNP-AR185-T2A-EGFP- Poly(A)-Right ITR</strong> |
| | </p> | | </p> |
| | </div> | | </div> |
| − | <h3 class="inner-h">How C185 works</h3> | + | <h3 class="inner-h">How it works</h3> |
| | <p class="inner-text"> | | <p class="inner-text"> |
| − | As has shown previously, BNP promoter activity is related to the severity of heart failure. So we want to use ET-1 and AngⅡ, two stimulus factor, to imitate the environment of heart failure and induce BNP promoter expression.To our dismay, BNP promoter didn’t response well to HF-related factors AngⅡand ET-1 in our vitro cell experiments, and its function remains to be investigated further.However, the composite part, C185, which is used to package AAV9 containing our therapeutic gene AR185 driven by CMV promoter, has shown the successful results. We have tested and results suggested that AR185 was effective and there were significant differences between AR185 group and control group. Then, the later sequence of the part, AR185, also can be expressed and fuction what it could specifically bind to RyR2 in rat cardiomyocytes and have the ability to inhibit PKA dependent S2808 phosphorylation in vitro. | + | As has shown previously, BNP promoter activity is related to the severity of heart failure. So we want to use ET-1 and AngⅡ, two stimulus factor, to imitate the environment of heart failure and induce BNP promoter expression. To our dismay, BNP promoter didn't response well to HF-related factors AngⅡand ET-1 in our vitro cell experiments, and its function remains to be investigated further. However, the composite part, C185, which is used to package AAV9 containing our therapeutic gene AR185 driven by CMV promoter, has shown the successful results. We have tested and results suggested that AR185 was effective and there were significant differences between AR185 group and control group. Then, the later sequence of the part, AR185, also can be expressed and fuction what it could specifically bind to RyR2 in rat cardiomyocytes and have the ability to inhibit PKA dependent S2808 phosphorylation in vitro. |
| | </p> | | </p> |
| − | <div style="text-align:center" class="resultimage">
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| − | <img src="https://static.igem.org/mediawiki/2018/a/a1/T--SMMU-China--Part_Collection_Pic_1.jpg" style="width: 60%;">
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| − | <p style="font-style: italic;text-align: center;padding: 0em 100px 1em;">
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| − | <strong>Figure 2. Design and Function of part of Left ITR-CMV-AR185-Poly(A)-Right ITR</strong>
| |
| − | </p>
| |
| − | </div>
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| | <h3 class="inner-h">See More</h3> | | <h3 class="inner-h">See More</h3> |
| | <p class="inner-text"> | | <p class="inner-text"> |
| | Integrated information about AR185-T2A-EGFP and BNP promotor posted on Demonstrate page. Or you can alternatively check the the (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2865013" target="_Blank">BBa_K2865013</a>) on the Registry of Standard Biological Parts website. | | Integrated information about AR185-T2A-EGFP and BNP promotor posted on Demonstrate page. Or you can alternatively check the the (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2865013" target="_Blank">BBa_K2865013</a>) on the Registry of Standard Biological Parts website. |
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| | </p> | | </p> |
| | <p class="inner-text"> | | <p class="inner-text"> |
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| | </table> | | </table> |
| | </center> | | </center> |
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| − | <h3 class="inner-h">References</h3>
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| − | <ol>
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| − | <li>Sergeeva, I.A. and V.M. Christoffels, Regulation of expression of atrial and brain natriuretic peptide, biomarkers for heart development and disease. Biochim Biophys Acta, 2013. 1832(12): p. 2403-13.</li>
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| − | <li>Bingham, A.J., et al., The repressor element 1-silencing transcription factor regulates heart-specific gene expression using multiple chromatin-modifying complexes. Mol Cell Biol, 2007. 27(11): p. 4082-92.</li>
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| − | <li>Kerkela, R., et al., Key roles of endothelin-1 and p38 MAPK in the regulation of atrial stretch response. Am J Physiol Regul Integr Comp Physiol, 2011. 300(1): p. R140-9.</li>
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| − | <li>Lu, Y.M., et al., DY-9760e inhibits endothelin-1-induced cardiomyocyte hypertrophy through inhibition of CaMKII and ERK activities. Cardiovasc Ther, 2009. 27(1): p. 17-27.</li>
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| − | </ol>
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| | </article> | | </article> |
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