Difference between revisions of "Part:BBa K404243"
 
(4 intermediate revisions by 3 users not shown)
Line 1: Line 1:
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K404243 short</partinfo>
 
<partinfo>BBa_K404243 short</partinfo>
−
<br><b>The RGD integrin binding motif, inserted into the 453 loop of [[Part:BBa_K404002|[AAV2]-Rep-VP123_p5-TATAless]]</b>
+
<br><b>The RGD integrin binding motif, inserted into the 453 loop of [[Part:BBa_K404007|pCMV_[AAV2]-VP123]]</b>
 +
{| style="color:black" cellpadding="6" cellspacing="1" border="2" align="left"
 +
! colspan="2" style="background:#66bbff;"|[https://parts.igem.org/Part:BBa_K404243 [pCMV_[AAV2]-VP123ex(ViralBrick-453-RGD)]
 +
|-
 +
|'''BioBrick Nr.'''
 +
|[https://parts.igem.org/Part:BBa_K404243 BBa_K404243]
 +
|-
 +
|'''RFC standard'''
 +
|[https://parts.igem.org/Help:Assembly_standard_10 RFC 10]
 +
|-
 +
|'''Requirement'''
 +
|pSB1C3<br>
 +
|-
 +
|'''Source'''
 +
|pAAV_MCS provided by Stratagene
 +
|-
 +
|'''Submitted by'''
 +
|[http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010]
 +
|}
 +
<br /><br /><br /><br /><br /><br /><br /><br />
 +
 
  
 
[[Image:Freiburg10_pCMV_VP123 453-RGD.png|thumb|center|480px]]<br>
 
[[Image:Freiburg10_pCMV_VP123 453-RGD.png|thumb|center|480px]]<br>
 +
 +
The AAV capsid consists of 60 capsid protein subunits. The three cap proteins VP1, VP2, and VP3 are encoded in an overlapping reading frame. Arranged in a stoichiometric ratio of 1:1:10, they form an icosahedral symmetry. The mRNA encoding for the cap proteins is transcribed from p40 and alternative spliced to minor and major products. Alternative splicing and translation initiation of VP2 at a nonconventional ACG initiation codon promote the expression of VP1, VP2 and VP3. The VP proteins share a common C terminus and stop codon, but begin with a different start codon. The N termini of VP1 and VP2 play important roles in infection and contain motifs that are highly homologous to the phospholipase A2 (PLA2) domain and nuclear localization signals (BR)(+).
 +
<br />
 +
<br>
 +
CMV promoter is derived from human Cytomegalovirus, which belongs to Herpesvirus group. All family members share the ability to remain in latent stage in the human body. CMV is located upstream of immediate-early gene. However, CMV promoter is an example of widely used promoters and is present in mammalian expression vectors. The advantage of CMV is the high-level constitutive expression in mostly all human tissues [Fitzsimons et al., 2002].
 +
<br>
  
 
===Usage and Biology===
 
===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.
 
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.
 +
 +
https://static.igem.org/mediawiki/parts/f/f6/Freiburg10_ViralBrick_motif_RGD.png
 +
  
 
<!-- -->
 
<!-- -->

Latest revision as of 16:05, 13 January 2011

pCMV_[AAV2]VP123 (ViralBrick-453-RGD)
The RGD integrin binding motif, inserted into the 453 loop of pCMV_[AAV2]-VP123

[pCMV_[AAV2-VP123ex(ViralBrick-453-RGD)]
BioBrick Nr. BBa_K404243
RFC standard RFC 10
Requirement pSB1C3
Source pAAV_MCS provided by Stratagene
Submitted by [http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010]










Freiburg10 pCMV VP123 453-RGD.png

The AAV capsid consists of 60 capsid protein subunits. The three cap proteins VP1, VP2, and VP3 are encoded in an overlapping reading frame. Arranged in a stoichiometric ratio of 1:1:10, they form an icosahedral symmetry. The mRNA encoding for the cap proteins is transcribed from p40 and alternative spliced to minor and major products. Alternative splicing and translation initiation of VP2 at a nonconventional ACG initiation codon promote the expression of VP1, VP2 and VP3. The VP proteins share a common C terminus and stop codon, but begin with a different start codon. The N termini of VP1 and VP2 play important roles in infection and contain motifs that are highly homologous to the phospholipase A2 (PLA2) domain and nuclear localization signals (BR)(+).

CMV promoter is derived from human Cytomegalovirus, which belongs to Herpesvirus group. All family members share the ability to remain in latent stage in the human body. CMV is located upstream of immediate-early gene. However, CMV promoter is an example of widely used promoters and is present in mammalian expression vectors. The advantage of CMV is the high-level constitutive expression in mostly all human tissues [Fitzsimons et al., 2002].

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.

Freiburg10_ViralBrick_motif_RGD.png


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 2429
    Illegal XhoI site found at 698
    Illegal XhoI site found at 884
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 665
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2955
    Illegal SapI site found at 1833