Collection-Lentiviral Vectors

LENTIVIRAL VECTORS:

Duke University Viral Vector Core plasmids: Control optogenetic and other constructs (no MTA required, unless specified otherwise). Publications should acknowledge the donor of the reagent and the Viral Vector Core at Duke University.

 

Reporter constructs & cassettes for cloning

  1. pLV-empty cassette with polylinker
  2. pLV-CMV-eGFP
  3. pLV-CMV-mCherry
  4. pLV-CMV- with a polylinker for fusion protein-eGFP
  5. pLV-CMV-eGFP-polylinker for cloning IRES, cleavage peptides, or miR/shRNA
  6. pLV-CMV-GFP-IRES-PURO
  7. pLV-CMV-GFP-Puro
  8. pLV-CMV-IRES-eGFP with a polylinker for cloning cleavage peptides, or miR/shRNA
  9. pLV-CMV-IRES-Luc with a polylinker for cloning cleavage peptides, or miR/shRNA
  10. pLV-hUbC-EGFP (Addgene 14883)

 Note: This is just a small portion of LV-vector backbones available from our core facility; the core recently developed a collection of empty, GFP/cherry, p2A, t2A, IRES vectors ready-for cloning of bicistronic transgenes with different promoters – email to viral.core@duke.edu or directly to the VVC director:  boris.kantor@duke.edu to inquire about our collection. Please note: all vector backbones are optimized and checked for packaging and expression.

 

 

Naïve/control constructs for shRNA

  1. pLV-U6-shRNA-negative control WPRE- Puro
  2. pLV-U6-shRNA-negative control WRE- eGFP
  3. pLV-CMV-eGFP-miR flank-shRNA-Luc-flank-miR- EM7-blast
  4. pLV-CMV-tGFP-IRES-Puro-shRNA negative control- miR flank-shRNA negative control- flank-miR
  5. pLV-mPGK-tGFP-IRES-Puro-shRNA negative control- miR flank-shRNA negative control- flank-miR
  6. pLV-CMV-IRES-eGFP- with a polylinker for cloning cleavage peptides, or miR/shRNA
  7. pLV-CMV-IRES-Luc- with a polylinker for cloning cleavage peptides, or miR/shRNA

 

Inducible systems

  1. pLV-CMV rtTA3-Blast (Addgene #26429)
  2. pLV –CMV-TetR Blast (Addgene #17492)
  3. pInducer20 (Tet-inducible; Addgene #44012)
  4. pLV-CMV-CRE (Addgene #12238)
  5. pLV-CMV-SV40-nls-CRE (Addgene #12265)

 

Optogenetic constructs

Dr. Karl Deisseroth’s lab constructs

For first time order of these vectors you will have to fill an MTA form with Dr. Deisseroth. To obtain MTA, please please contact Dr. Karl Deisseroth and Charu Ramakrishnan. Please upload a copy of your MTA when placing your online order.

Contact email of Charu Ramakrishnan: charur@stanford.edu

Contact email of Dr. Karl Deisseroth: deissero@stanford.edu

 

  1. pLenti-CaMKIIa-eArch 3.0-eYFP
  2. pLenti-CaMKIIa-eArchT 3.0-eYFP
  3. pLenti-CaMKIIa-eMac 3.0-eYFP
  4. pLenti-CaMKIIa-C1V1 (E122T)-TS-eYFP
  5. pLenti-CaMKIIa-C1V1 (E162T)-TS-eYFP
  6. pLenti-CaMKIIa-C1V1 (E122T/E162T)-TS-eYFP
  7. pLenti-CaMKIIa-hChR2 (C128S/D156A)-eYFP
  8. pLenti-CaMKIIa-hChR2(T159C)-eYFP-WPRE
  9. pLenti-CaMKIIa-hChR2(E123T/T159C)-eYFP-WPRE
  10. pLenti-EF1a-hChR2-eYFP-WPRE (a.k.a. pLECYT)
  11. pLenti-EF1a-hChR2(H134R)-eYFP-WPRE (a.k.a. pLECYT)
  12. pLenti-CaMKIIa-hChR2-mCherry-WPRE
  13. pLenti-CaMKIIa-hChR2-eYFP-WPRE
  14. pLenti-CaMKIIa-hChR2(H134R)-eYFP-WPRE
  15. pLenti-Synapsin-hChR2(H134R)-eYFP-WPRE
  16. pLenti-CaMKIIa-eNpHR 3.0-eYFP
  17. pLenti-hSyn-eNpHR3.0-eYFP

 

Optical Excitation: Volvox Channelrhodopsin-1 (VChR1)

  1. Available in a standard mammalian expression vector and lentivirus vectors
  2. 1/VChR1-eYFP
  3. 1/VChR1-mCherry
  4. pLenti-CaMKIIa-VChR1-eYFP-WPRE
  5. pLenti-CaMKIIa-VChR1-mCherry-WPRE

For more info see:

A gene-fusion strategy for stoichiometric and co-localized expression of light-gated membrane proteins.

Color-tuned channelrhodopsins for multiwavelength ontogenetic.

 

Maps of the constructs are available here http://web.stanford.edu/group/dlab/optogenetics/sequence_info.html

 

 

*The VChR1 gene is codon-optimized for mammalian expression.

 

 

Optical Inhibition: Halorhodopsin (NpHR), Third-generation Optogenetic Inhibition

Arch 3.0 ArchT 3.0, and Mac 3.0

For more info see: Amygdala circuitry mediating reversible and bidirectional control of anxiety.

 

Second (2.0) and Third (3.0) generations of NpHR are available. eNpHR2.0 may be superior in some cell types, including photoreceptors, see: Genetic reactivation of cone photoreceptors restores visual responses in retinitis pigmentosa.

 

 

  1. pLenti- hSyn- eNpHR 3.0- eYFP
  2. pLenti-CaMKIIa-eNpHR2.0-eYFP
  3. pLenti- CaMKIIa- eArchT3.0- eYFP
  4. pLenti- CaMKIIa- eArch3.0- eYFP
  5. pLenti- CaMKIIa- eMac3.0- eYFP

 

 

Optical Excitation: Channelrhodopsin-2 (ChR2)

For more info see:

Channelrhodopsin-2 and optical control of excitable cells.

Circuit-breakers: optical technologies for probing neural signals and systems.

 

  1. pLenti- CaMKIIa- hChR2-eYFP-WPRE (a.k.a. pLECYT)
  2. pLenti-EF1a-hChR2-eYFP-WPRE (a.k.a. pLECYT)
  3. pLenti-EF1a-hChR2(H134R)-eYFP-WPRE (a.k.a. pLECYT)
  4. pLenti-CaMKIIa-hChR2-mCherry-WPRE
  5. pLenti-CaMKIIa-hChR2-eYFP-WPRE
  6. pLenti-Hcrt-hChR2(H134R)-eYFP
  7. pLenti-Synapsin-hChR2(H134R)-eYFP-WPRE
  8. pLenti-mThy1-hChR2(H134R)-eYFP
  9. pLenti-hThy1-hChR2(H134R)-eYFP

 

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. For more info see: Bi-stable neural state switches.

 

  1. pLenti-CaMKIIa-hChR2(C128A)-eYFP
  2. pLenti-CaMKIIa-hChR2(C128S)-eYFP
  3. pLenti-CaMKIIa-hChR2(C128T)-eYFP

 

Ultrafast Optogenetic Control

 

For more info see: Ultrafast optogenetic control.

  1. pLenti-CaMKIIa-hChR2(E123T-H134R)-eYFP
  2. pLenti-CaMKIIa-hChR2(T159C)-eYFP-WPRE
  3. pLenti-CaMKIIa-hChR2(E123T/T159C)-eYFP

 

Stabilized Step Function Opsins SSFO

Newly developed ChR2 variants which act to stabilize the conducting state of the channel to deactivate with a time constant of nearly 30 minutes following a brief pulse of activating blue light. They can be deactivated by yellow light (590nm). The stabilized open state of the channel allows for both lower power activation, meaning in some circumstances the light delivery system need not penetrate the brain, as well as for behavior in the absence of a tethered laser or other light delivery system.

  1. pLenti-CaMKIIa-hChR2 (C128S/D156A)-eYFP

 

 

 

Red-Shifted Optical Excitation: C1V1 variants C1V1

For more info see: Two-photon optogenetics of dendritic spines and neural circuits.

Simultaneous cellular-resolution optical perturbation and imaging of place cell firing fields.

 

  1. pLenti-CaMKIIa-C1V1-eYFP
  2. pLenti-CaMKIIa-C1V1-TS-eYFP
  3. pLenti-CaMKIIa-C1V1 (E122T)-TS-eYFP
  4. pLenti-CaMKIIa-C1V1 (E162T)-TS-eYFP
  5. pLenti-CaMKIIa-C1V1 (E122T/E162T)-TS-eYFP

 

 

Dr. Edward Boyden lab constructs

 

All LV constructs obtained from Addgene: No MTA required unless used for commercial purposes

 

Neural Silencers/Activators

For more info see: Millisecond-timescale, genetically targeted optical control of neural activity.

Prosthetic systems for therapeutic optical activation and silencing of genetically-targeted neurons.

A gene-fusion strategy for stoichiometric and co-localized expression of light-gated membrane .

 

  1. pLenti-CaMKIIa-ChR2-GFP
  2. FCK-Halo-GFP pLenti-CaMKIIa – codon-optimized halorhodopsin codon-optimized halorhodopsin-GFP
  3. pLenti-CaMKIIa – codon-optimized halorhodopsin Arch-GFP
  4. ChR2-GFP-2A-Halo-YFP pLenti-ChR2-GFP-2A-Halo-eYFP
  5. FCK-Mac-GFP pLenti-CaMKIIa – codon-optimized halorhodopsinMac-GFP
  6. Fubi-ChR2-GFP pLenti-Fubi-ChR2-GFP
  7. FCK-Arch-CFP pLenti- CaMKIIa -Arch-CFP
  8. pLenti- CaMKIIa -ArchT-CFP
  9. FCK-ss-Prl-Arch-GFP pLenti- CaMKIIa-ss-Prl-Arch-GFP
  10. pLenti- CaMKIIa- ChrimsonR-GFP

Dr. Adam Cohen lab constructs

All LV constructs obtained from Addgene: No MTA required unless used for commercial purposes

DRH337: AAV-hsyn-CheRiff-eGFPArchaerhodopsin-based voltage indicators; Channelrhodopsin actuator

For more info see: All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins.

 

  1. pLenti-DRH296: FCK-Optopatch2 #51694 Addgene
  2. DRH229: FCK-QuasAr1-mO2 DRH229: FCK-QuasAr1-mO2pLenti-DRH229: FCK-QuasAr1-mO2 #51629 Addgene
  3. DRH334: FCK-QuasAr2-mO2pLenti-DRH334: FCK-QuasAr2-mO2 #51692 Addgene
  4. DRH313: FCK-CheRiff-eGFPpLenti-DRH313: FCK-CheRiff-Egfp #51693 Addgene
  5. DRH335: FCK-Optopatch1pLenti-DRH335: FCK-Optopatch1 #51695 Addgene
  6. pLenti-Arch(D95H)-eGFP #34616 Addgene
  7. pLenti-FCK-QuasAr2-Citrine #59172 Addgene
  8. FCK-QuasAr2-mRuby2 eFRETpLenti-FCK-QuasAr2-mRuby2 eFRET #59174 Addgene
  9. CK-QuasAr2-mRuby2 eFRET FCK-QuasAr2-mOrange2 eFRETpLenti-FCK-QuasAr2-mOrange2 eFRET #59173 Addgene
  10. Lenti-MOS010: pLX304-CheRiff-GFP #62986 Addgene

Calcium sensors/indicators

91.   pLenti-pCamKII-GCaMP6f- WPRE

92.   pLenti-CMV-GCaMP6f- WPRE

93.   pLenti-CAG-GCaMP6f- WPRE

94.  pLenti-CMV-QuasAr2-TS-GCaMP6f #72303 Addgene

95.  pLenti-pCamKII-QuasAr2-TS-GCaMP6f #72304 Addgene

96.  pHAGE-RSV-tdTomato-2A-GCaMP6s #80316 Addgene

97.  pHAGE-RSV-GCaMP6s #80146  Addgene