SEQUENCE INFO

  Note: Due to ease of recombination, AAV and lentivirus vectors should be amplified in a recombination deficient bacteria strain such as Invitrogen's OneShot Stbl3 cells.
   
   
  Optical Control of Intracellular Signaling: Opto-XRs
     
 

Chimeric fusions of bovine Rhodopsin and adrenergic G-Protein Coupled Receptors allowing optical control of GPCR signaling cascades. Proteins are activated by 500nm light.
 
pcDNA3.1v5his-opto-a1AR-EYFP [ Vector Map ]
pcDNA3.1v5his-opto-b2AR-EYFP [ Vector Map ]
     
     
  Bi-stable excitation: Step Function Opsins (SFOs)
     
 

Three point-mutants of humanized ChR2 convert a brief pulse of light into a stable step in membrane potential. The lentiviral vectors were created by site-directed mutagenesis of the C128 position in ChR2. All three mutants are activated by blue (470nm) light. Photocurrents generated by ChR2(C128A) and ChR2(C128S) can be effectively terminated by a pulse of green (542nm) light.

 
pLenti-CaMKIIa-hChR2(C128A)-EYFP-WPRE [ Vector Map ]
pLenti-CaMKIIa-hChR2(C128S)-EYFP-WPRE [ Vector Map ]
pLenti-CaMKIIa-hChR2(C128T)-EYFP-WPRE [ Vector Map ]
     
     
  Cre-inducible Adeno-associated Virus: DIO-AAV  
     
  For cell type-specific targeting and to capitalize on the large number of Cre driver lines with the flexible virus injection/fiberoptic approach, we have developed a tool we call DIO-AAV (doublefloxed inverse orf) AAV.
     
 
     
pAAV-double floxed-hChR2(H134R)-EYFP-WPRE-pA [ Vector Map ]
pAAV-double floxed-hChR2(H134R)-mCherry-WPRE-pA [ Vector Map ]
pAAV-double floxed-eNpHR-EYFP-WPRE-pA [ Vector Map ]
   
   
  Optical Excitation: Channelrhodopsin-2 (ChR2)
     
 

There are two versions of the ChR2 sequence, one containing the wildtype sequence and another containing condons optimized for mammalian expression (hChR2). In-frame fusions to mCherry or EYFP are available to make visualization of ChR2-expressing cells easier. ChR2 and XFP are fused via a NotI site. The linker is GCGGCCGCC.
     
  ChR2-XFPs are available either in a standard mammalian expression vector containing the CMV promoter or in lentiviral expression vectors under the control of the ubiquitous EF-1a or the neuron-specific CaMKIIa or human Synapsin I promoters. The structure of the lentivirus is shown below.
 
pcDNA3.1/hChR2-mCherry [ Vector Map ]
pcDNA3.1/hChR2-EYFP [ Vector Map ]
pcDNA3.1/hChR2(H134R)-EYFP [ Vector Map ]
pLenti-EF1a-hChR2-EYFP-WPRE (a.k.a. pLECYT) [ Vector Map ]
pLenti-EF1a-hChR2(H134R)-EYFP-WPRE (a.k.a. pLECYT) [ Vector Map ]
  pLenti-CaMKIIa-hChR2-mCherry-WPRE [ Vector Map ]
  pLenti-CaMKIIa-hChR2-EYFP-WPRE [ Vector Map ]
  pLenti-Synapsin-hChR2(H134R)-EYFP-WPRE [ Vector Map ]
     
     
  Optical Inhibition: Halorhodopsin (NpHR)  
     
  The NpHR sequence here has been optimized for mammalian expression. The NpHR-EYFP inframe fusion genes are made via a NotI site with the linker GCGGCCGCC. The start codon on EYFP has been deliberately removed. To reduce membrane blebbing or other toxicity at high levels of expression, we have generated a modified eNpHR by adding signaling peptides to enhance membrane translocation and ER export.
     
  Currently, the only version of halorhodopsin that is available for shipping is pLenti-CaMKII-eNpHR-EYFP.
 
Lenti-CaMKIIa-eNpHR-EYFP-WPRE [ Vector Map ]
   
   
   
  Optical Excitation: Volvox Channelrhodopsin-1 (VChR1)  
     
  The VChR1 sequence here has been optimized for mammalian expression. The VChR1-EYFP inframe fusion genes are made via a NotI site with the linker GCGGCCGCC. The start codon on EYFP has been deliberately removed. VChR1-EYFP are also available in a standard mammalian expression vector and lentivirus vectors.
 
pcDNA3.1/VChR1-EYFP [ Vector Map ]
pcDNA3.1/VChR1-mCherry [ Vector Map ]
Lenti-CaMKIIa-VChR1-EYFP-WPRE [ Vector Map ]
Lenti-CaMKIIa-VChR1-mCherry-WPRE [ Vector Map ]