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REPORTERS OFFERED FOR SERVICES AND LICENSE
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FUNCTIONAL REPORTER CELL LINES AND CONSTRUCTS  
   
REAL TIME IN VIVO IMAGING OF APOPTOSIS
  
 

MIR's caspase activation assays generate in vivo bioluminescence or fluorescence-based readouts of apoptosis. These assays yield real time in vivo imaging of drug function, requiring fewer animals and produces tighter correlations than excision based immunologic readouts. Bioluminescence imaging increase both detection sensitivity and imaging throughput. MIR is offering this technology for license as well as a contract research service. This technology is available exclusively through MIR.

 
   
For this construct, luciferase is bound to a sequestering protein (estrogen receptor) by a DEVD peptide linker. While luciferase is bound to the sequestering protein, luciferase is inactive. Caspase 3 is involved in both the extrinsic and intrinsic apoptotic pathways. Caspase 3, when active, cleaves the DEVD tether, releasing luciferase from the sequestering protein. Active luciferase can then act on its substrate and release a bioluminescent signal, indicating apoptosis.
How does this technology work?
For this construct, lac z (a fluorescent molecule) is bound to a sequestering protein (estrogen receptor) by a DEVD peptide linker. While lac z is bound to the sequestering protein, lac z is unable to fluoresce. Caspase 3 is involved in both the extrinsic and intrinsic apoptotic pathways. Caspase 3, when active, cleaves the DEVD tether, releasing lac z from the sequestering protein. Unbound lac z can fluoresce when excited at the right wavelength, indicating apoptosis.

The left figure depicts cells are stably transfected with a luciferase construct that is fused with ER. The ER silences luciferase activity due to sequestration. Activation of caspase 3 during apoptosis results in cleavage at DEVD site releasing luciferase. Free luciferase can now (in the presence of luciferin) generate bioluminescence which can be imaged in vitro and in vivo using a Xenogen imaging system. The left figure depicts cells can stably transfected with a lac Z construct that is fused with ER that silences lac Z. Activation of caspase 3 during apoptosis results in cleavage at DEVD site releasing lac Z which can now (in the presence of DDAOG) generate fluorescence which can be imaged in vitro and in vivo using a Xenogen imaging system.
 
   

Caspase activation in luciferase based assay correlated with increased bioluminescence

Caspase activation in Lac-z based assay correlated with increased fluorescence

Caspase activation in luciferase based assay correlated with increased bioluminescence
 
 
CONSTITUITIVE REPORTER MODELS  
   

MIR has a number of cell lines that constitutively express luciferase. These models are well suited for orthotopic and metastatic studies. Below is a list of luciferase producing cell lines that MIR offers for services:

  • B16-luc

  • PC3-luc

  • MDA-MB-231-luc

  • SK-OV-3-luc

Other cell lines are in the process of being validated. Please contact MIR for more information on these upcoming models.

 
 
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Site map for www.molecularimaging.com
800 Technology Drive• Ann Arbor, Michigan 48108
Phone: 734.821.1063 Fax: 734.821.1066
  
 
updated:  3/18/08