Difference between revisions of "Part:BBa K404212"
| (3 intermediate revisions by one other user not shown) | |||
| Line 1: | Line 1: | ||
__NOTOC__ | __NOTOC__ | ||
<partinfo>BBa_K404212 short</partinfo> | <partinfo>BBa_K404212 short</partinfo> | ||
| + | {| style="color:black" cellpadding="6" cellspacing="1" border="2" align="left" | ||
| + | ! colspan="2" style="background:#66bbff;"|[https://parts.igem.org/Part:BBa_K404212 ViralBrick-587KO-RGD] | ||
| + | |- | ||
| + | |'''BioBrick Nr.''' | ||
| + | |[https://parts.igem.org/Part:BBa_K404212 BBa_K404212] | ||
| + | |- | ||
| + | |'''RFC standard''' | ||
| + | |[https://parts.igem.org/Help:Assembly_standard_25 RFC 25] | ||
| + | |- | ||
| + | |'''Requirement''' | ||
| + | |pSB1C3<br> | ||
| + | |- | ||
| + | |'''Source''' | ||
| + | | | ||
| + | |- | ||
| + | |'''Submitted by''' | ||
| + | |[http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010] | ||
| + | |} | ||
| + | <br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/> | ||
<br><b>The RGD integrin binding motif including a knockout of the natural HSPG tropism, ready for insertion into the 587 loop</b> | <br><b>The RGD integrin binding motif including a knockout of the natural HSPG tropism, ready for insertion into the 587 loop</b> | ||
| Line 6: | Line 25: | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
| + | <div style="width:965px; height:400px; "> | ||
| + | <div style="float:left; width:460px; height:auto; margin: 0px 5px 0px 5px; text-align:justify;"> | ||
| + | |||
Integrins are transmembrane proteins that, among other functions, mediate cell attachment to surrounding tissues. They bind to a motif consisting of the amino acids arginine, glycine and aspartic acid (RGD in one-letter code). Because Integrin is highly expressed in many tumor cell lines (Albelda et al., 1990), (Damjanovich, Albelda, Mette, & Buck, 1992), (Lessey et al., 1995), (Smythe, LeBel, Bavaria, Kaiser, & Albelda, 1995), (Gladson & Cheresh, 1991), AAV particles displaying the RGD motif on various positions in their capsid proteins have been created by (Shi et al., 2003). Particles displaying RGD at amino acid positions 584 & 588 as well as 453 or 587 (Boucas et al., 2009) showed transduction efficiencies similar to wt AAV, even when the cells’ HSPG receptors were blocked by heparin sulfate or when the natural HSPG binding motif on the capsid surface was knocked out. To further broaden the area of therapeutic application, we created a ViralBrick containing the RGD motive to specifically target cells with low HSPG-/high Integrin expression.<br> | Integrins are transmembrane proteins that, among other functions, mediate cell attachment to surrounding tissues. They bind to a motif consisting of the amino acids arginine, glycine and aspartic acid (RGD in one-letter code). Because Integrin is highly expressed in many tumor cell lines (Albelda et al., 1990), (Damjanovich, Albelda, Mette, & Buck, 1992), (Lessey et al., 1995), (Smythe, LeBel, Bavaria, Kaiser, & Albelda, 1995), (Gladson & Cheresh, 1991), AAV particles displaying the RGD motif on various positions in their capsid proteins have been created by (Shi et al., 2003). Particles displaying RGD at amino acid positions 584 & 588 as well as 453 or 587 (Boucas et al., 2009) showed transduction efficiencies similar to wt AAV, even when the cells’ HSPG receptors were blocked by heparin sulfate or when the natural HSPG binding motif on the capsid surface was knocked out. To further broaden the area of therapeutic application, we created a ViralBrick containing the RGD motive to specifically target cells with low HSPG-/high Integrin expression.<br> | ||
| − | + | </div> | |
| + | |||
| + | <html><div style="float:right; width:480px; height:auto; "><img src="https://static.igem.org/mediawiki/parts/f/f6/Freiburg10_ViralBrick_motif_RGD.png" width="460" | ||
| + | height="auto"/></div></div></html> | ||
| + | ===Restriction sites=== | ||
<table border="1" style="float:right; width:480px; height:auto;> | <table border="1" style="float:right; width:480px; height:auto;> | ||
<tr> | <tr> | ||
| Line 34: | Line 60: | ||
<tr> | <tr> | ||
</table> | </table> | ||
| − | |||
===Results=== | ===Results=== | ||
Latest revision as of 02:28, 28 October 2010
ViralBrick-587KO-RGD
| ViralBrick-587KO-RGD | |
|---|---|
| BioBrick Nr. | BBa_K404212 |
| RFC standard | RFC 25 |
| Requirement | pSB1C3 |
| Source | |
| Submitted by | [http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010] |
The RGD integrin binding motif including a knockout of the natural HSPG tropism, ready for insertion into the 587 loop
Usage and Biology
Integrins are transmembrane proteins that, among other functions, mediate cell attachment to surrounding tissues. They bind to a motif consisting of the amino acids arginine, glycine and aspartic acid (RGD in one-letter code). Because Integrin is highly expressed in many tumor cell lines (Albelda et al., 1990), (Damjanovich, Albelda, Mette, & Buck, 1992), (Lessey et al., 1995), (Smythe, LeBel, Bavaria, Kaiser, & Albelda, 1995), (Gladson & Cheresh, 1991), AAV particles displaying the RGD motif on various positions in their capsid proteins have been created by (Shi et al., 2003). Particles displaying RGD at amino acid positions 584 & 588 as well as 453 or 587 (Boucas et al., 2009) showed transduction efficiencies similar to wt AAV, even when the cells’ HSPG receptors were blocked by heparin sulfate or when the natural HSPG binding motif on the capsid surface was knocked out. To further broaden the area of therapeutic application, we created a ViralBrick containing the RGD motive to specifically target cells with low HSPG-/high Integrin expression.
Restriction sites
| Restriction site | Enzyme | Recognition sequence | Activity | Temp. |
|---|---|---|---|---|
| upstream 587 | [http://www.neb.com/nebecomm/products/productR3136.asp BamHI-HF] |  |
100;50;10;100 | 37°C |
| downstream 587 | [http://www.neb.com/nebecomm/products/productR3151.asp PvuII-HF] |  |
0;25;0;100 | 37°C |
Results
We could show a differential targeting towards tumor cells overexpressing integrin on their surface using AAV particles containing the RGD loop insertion with knocked out HSPG tropism. Titers were determined via qPCR (for details, see [http://2010.igem.org/Team:Freiburg_Bioware/Project/Results#targeting team wiki])
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 10
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]