Difference between revisions of "Part:BBa K404212"

Revision as of 17:22, 26 October 2010

ViralBrick-587KO-RGD
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 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:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 10
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 ===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>
-[[Image:Freiburg10_ViralBrick_motif_RGD.png|thumb|center|480px]]
+</div>
+<html><div style="float:right; width:480px; height:auto; "><img src="https://static.igem.org/mediawiki/parts/5/59/Freiburg10_ViralBrick_motif_RGD.png" width="460"
+height="auto"/></div></div><br><br><br><br></html>
 <table border="1" style="float:right; width:480px; height:auto;>